Methods for altering cell fate

ABSTRACT

The invention provides methods for altering the expression profile of a cell to convert the cell from one cell type to a desired cell type. These reprogrammed cells may be used in a variety of medical applications for treating a mammal in need of a particular cell type.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/015,824, filed Dec. 10, 2001 now abandoned, which claims priorityfrom U.S. provisional application 60/258,152, filed Dec. 22, 2000, eachof which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

In general, the invention features methods for converting cells into adesired cell type and methods for administering these reprogrammed cellsto a mammal for the treatment or prevention of disease.

Despite having essentially the same genome, different classes of somaticcells in a particular mammal have distinctive phenotypes due to thedifferent combinations of genes that they express. These differentexpression profiles allow cells to perform certain functions, such asthe secretion of a hormone or cartilage.

Because many diseases and injuries are caused by damage to a particularclass of cells, methods are needed to produce cells of a desired celltype that may be used to replace these damaged cells. Preferably, thesereplacement cells have the same genotype as the damaged cells.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide methods for alteringthe characteristics or functions of cells. In particular, these methodsinvolve incubating a nucleus or chromatin mass from a donor cell with areprogramming media (e.g., a cell extract) under conditions that allownuclear or cytoplasmic components such as transcription factors to beadded to, or removed from, the nucleus or chromatin mass. Preferably,the added transcription factors promote the expression of mRNA orprotein molecules found in cells of the desired cell type, and theremoval of transcription factors that would otherwise promote expressionof mRNA or protein molecules found in the donor cell. If desired, thechromatin mass may then be incubated in an interphase reprogrammingmedia (e.g., an interphase cell extract) to reform a nucleus thatincorporates desired factors from either reprogramming media. Then, thenucleus or chromatin mass is inserted into a recipient cell orcytoplast, forming a reprogrammed cell of the desired cell type. In arelated method, a permeabilized cell is incubated with a reprogrammingmedia (e.g., a cell extract) to allow the addition or removal of factorsfrom the cell, and then the plasma membrane of the permeabilized cell isresealed to enclose the desired factors and restore the membraneintegrity of the cell. If desired, the steps of any of these methods maybe repeated one or more times or different reprogramming methods may beperformed sequentially to increase the extent of reprogramming,resulting in a greater alteration of the mRNA and protein expressionprofile in the reprogrammed cell. Furthermore, reprogramming medias maybe made representing combinations of cell functions (e.g., mediascontaining extracts or factors from multiple cell types) to produceunique reprogrammed cells possessing characteristics of multiple celltypes.

Accordingly, in a first aspect, the invention provides a method ofreprogramming a cell. This method involves incubating a nucleus with areprogramming media (e.g., a cell extract) under conditions that allowthe removal of a factor from the nucleus or the addition of a factor tothe nucleus. Then the nucleus or a chromatin mass formed from incubationof the nucleus in the reprogramming media is inserted into a recipientcell or cytoplast, thereby forming a reprogrammed cell. In one preferredembodiment, the nucleus is incubated with an interphase reprogrammingmedia (e.g., an interphase cell extract). Preferably, the nucleusremains membrane-bounded, and the chromosomes in the nucleus do notcondense during incubation with this interphase reprogramming media. Inanother preferred embodiment, a chromatin mass is formed from incubationof the nucleus in a mitotic reprogramming media (e.g., a mitoticextract). Preferably, this chromatin mass is then incubated in aninterphase reprogramming media under conditions that allow a nucleus toreform, and the reformed nucleus is inserted into the recipient cell orcytoplast.

In a related aspect, the invention provides another method ofreprogramming a cell. This method involves incubating a chromatin masswith a reprogramming media (e.g., a cell extract) under conditions thatallow the removal of a factor from the chromatin mass or the addition ofa factor to the chromatin mass. Then the chromatin mass or nucleusformed from incubation of the chromatin mass in a reprogramming media(e.g., an interphase extract) is inserted into a recipient cell orcytoplast, thereby forming a reprogrammed cell. In one preferredembodiment, the chromatin mass is generated by incubating a nucleus froma donor cell in a detergent and salt solution, in a protein kinasesolution, or in a mitotic reprogramming media in the presence or absenceof an antibody to NuMA or to another protein of the nucleus. In anotherpreferred embodiment, the chromatin mass is isolated from mitotic cells.

In another related aspect, the invention provides yet another method ofreprogramming a cell. This method involves incubating a permeabilizedcell with a reprogramming media (e.g., a cell extract) under conditionsthat allow the removal of a factor from the nucleus or chromatin mass ofthe permeabilized cell or the addition of a factor to the nucleus orchromatin mass, thereby forming a reprogrammed cell. In one preferredembodiment, the permeabilized cell is incubated with an interphasereprogramming media (e.g., an interphase cell extract). Preferably, thenucleus in the permeabilized cell remains membrane-bounded, and thechromosomes in the nucleus do not condense during incubation with thisinterphase reprogramming media. In another preferred embodiment, achromatin mass is formed from incubation of the permeabilized cell in amitotic reprogramming media. In yet another preferred embodiment, thereprogrammed cell is incubated under conditions that allow the membraneof the reprogrammed cell to reseal. If desired, the permeabilized cellmay be formed by incubating an intact cell with a detergent, such asdigitonin, or a bacterial toxin, such as Streptolysin O.

The invention also provides reprogrammed cells generated using anymethod of the invention or a combination of methods of the invention.These cells are useful for the treatment or prevention of a disease dueto a deficiency in a particular cell type. Additionally, reprogrammedcells that express two or more mRNA molecules or proteins that are eachspecific for a certain cell type may have novel combinations ofphenotypes and activities that are useful for the treatment of disease.For example, cells that maintain the ability of the donor cell to divideand gain the ability to form a functional T-cell receptor or afunctional neurofilament are useful for the generation of multipleT-cells or neurons for therapeutic applications. Once transplanted intoa subject, these cells may maintain the ability to divide, therebyreducing the dose or dosing frequency of the transplant cells that isrequired to treat, prevent, or stabilize a disease. The characterizationof these cells may also result in the identification of proteinsinvolved in the regulation of gene expression.

In one such aspect, the invention features a cell that expresses acombination of two or more endogenous mRNA molecules or endogenousproteins that is not expressed by a naturally-occurring cell. In arelated aspect, the invention features a cell that expresses acombination of two or more endogenous mRNA molecules or endogenousproteins at a level that is at least 10, 20, 50, 75, or 100 fold greaterthan the expression level of the corresponding mRNA molecules orproteins in any naturally-occurring cell. In preferred embodiments ofthe above aspects, the cell expresses a combination of 5, 10, 25, 50,75, 100, 150, 300, or more endogenous mRNA molecules or endogenousproteins that is not expressed by a naturally-occurring cell. In anotherpreferred embodiment, the cell expresses 1, 3, 5, 10, 25, 50, 100, ormore endogenous mRNA molecules or endogenous proteins that are specificfor one cell type and expresses 1, 3, 5, 10, 25, 50, 100, or moreendogenous mRNA molecules or endogenous proteins that are specific foranother cell type. In other preferred embodiments, the cell has acombination of 2, 5, 10, 25, 50, 75, 100, 150, 300, or more activitiesor phenotypes that are not exhibited in a naturally-occurring. In yetother preferred embodiments, the cell is able to divide or isimmortalized and expresses a neuronal protein such as the NF200neurofilament protein or any other protein expressed by differentiatedneurons. In still other preferred embodiments, the cell is able todivide or is immortalized and expresses IL-2, an IL-2 receptor, a T-cellreceptor, CD3, CD4 and CD8, CD45 tyrosine phosphatase, or any otherprotein expressed in hematopoietic cells. In yet another embodiment, thecell is formed from the reprogramming of a donor fibroblast cell,nucleus, or chromatin mass, and the reprogrammed cell expresses one ormore cytoskeleton proteins such as an integrin at a level that is atleast 25, 50, 75, 90, or 95% lower that the corresponding level in thedonor fibroblast under the same conditions. In another embodiment, areprogrammed cell formed from a donor fibroblast or liver cell (e.g., ahepatocyte) expresses IL-2, a neurofilament protein, a T-cell receptor,Oct4, or insulin.

In a related aspect, the invention provides a cell that expresses aT-cell specific protein (e.g., T-cell receptor protein, IL-2 receptor,CD3, CD4, or CD8) and one or more fibroblast-specific proteins.Preferably, stimulation of the cell with an antigen or an anti-CD3antibody induces the expression of the α-chain of the IL-2 receptor. Inanother aspect, the invention provides a cell that expresses ahematopoietic-specific protein (e.g., CD45 typrosine phosphatase) andone or more fibroblast-specific proteins. In another related aspect, theinvention provides a cell that expresses a neuron-specific protein(e.g., a neurofilament protein such as NF200) or forms neurites andexpresses one or more fibroblast-specific proteins. In still anotheraspect, the invention provides a cell that expresses a neurofilamentprotein (e.g., NF200) or forms neurites and is immortalized. In yetanother aspect, the invention provides a cell that expresses a stemcell-specific protein (e.g., Oct4) or alkaline phosphatase and one ormore fibroblast-specific proteins. In still another aspect, theinvention provides a cell that expresses one or more fibroblast-specificproteins and grows in aggregates, forms colonies, or forms embryoidbodies. Preferred fibroblast-specific proteins include cell adhesionmolecules that e.g., promote anchoring of one or more reprogrammed cellsto a site of interest in a host patient. Fibroblast-specific growthfactors (e.g., the FGF family of proteins) are other exemplaryfibroblast-specific proteins.

These methods for reprogramming cells are useful for the generation ofcells of a desired cell type, for example, for medical applications.Accordingly, the invention also provides methods for the treatment orprevention of disease in a mammal that include administering areprogrammed cell to the mammal.

In one such method, the invention features a procedure for treating orpreventing a disease, disorder, or condition in a mammal. This methodinvolves incubating a nucleus from a donor cell with a reprogrammingmedia (e.g., a cell extract) under conditions that allow the removal ofa factor from the nucleus or the addition of a factor to the nucleus.The nucleus or a chromatin mass formed from the nucleus is inserted intoa recipient cell or cytoplast, thereby forming a reprogrammed cell. Thereprogrammed cell is then administered to the mammal in need of the celltype. In one preferred embodiment, the nucleus is incubated with aninterphase reprogramming media. Preferably, the nucleus remainsmembrane-bounded, and the chromosomes in the nucleus do not condenseduring incubation with this interphase reprogramming media. In anotherpreferred embodiment, a chromatin mass is formed from incubation of thenucleus in a mitotic reprogramming media. Preferably, this chromatinmass is then incubated in an interphase reprogramming media underconditions that allow a nucleus to be formed from the chromatin mass,and the reformed nucleus is inserted into the recipient cell orcytoplast. Preferably, the donor cell is from the mammal (for example, ahuman) in need of the cell type. Examples of diseases, disorders, orconditions that may be treated or prevented include neurological,endocrine, structural, skeletal, vascular, urinary, digestive,integumentary, blood, immune, auto-immune, inflammatory, endocrine,kidney, bladder, cardiovascular, cancer, circulatory, digestive,hematopoeitic, and muscular diseases, disorders, and conditions. Inaddition, reprogrammed cells may be used for reconstructiveapplications, such as for repairing or replacing tissues or organs.

In a related aspect, the invention provides another method of treatingor preventing a disease, disorder, or condition in a mammal (forexample, a human). This method involves incubating a chromatin mass froma donor cell with a reprogramming media (e.g., a cell extract) underconditions that allow the removal of a factor from the chromatin mass orthe addition of a factor to the chromatin mass. The chromatin mass or anucleus formed from incubating the chromatin mass in an interphasereprogramming media is inserted into a recipient cell or cytoplast,thereby forming a reprogrammed cell. In one preferred embodiment, thechromatin mass used in this method is generated by incubating a nucleusfrom a donor cell in a detergent and salt solution, in a protein kinasesolution, or in a mitotic reprogramming media in the presence or absenceof an antibody to NuMA. In another preferred embodiment, the chromatinmass is isolated from mitotic cells. The reprogrammed cell is thenadministered to a mammal in need of the cell type. Preferably, the donorcell is from the recipient mammal. Examples of diseases, disorders, orconditions that may be treated or prevented include neurological,endocrine, structural, skeletal, vascular, urinary, digestive,integumentary, blood, immune, auto-immune, inflammatory, endocrine,kidney, bladder, cardiovascular, cancer, circulatory, digestive,hematopoeitic, and muscular diseases, disorders, and conditions. Inaddition, reprogrammed cells may be used for reconstructiveapplications, such as for repairing or replacing tissues or organs.

In still another related aspect, the invention provides another methodof treating or preventing a disease, disorder, or condition in a mammal(for example, a human) that involves incubating a permeabilized cellwith a reprogramming media (e.g., a cell extract) under conditions thatallow the removal of a factor from the nucleus or chromatin mass of thepermeabilized cell or the addition of a factor to the nucleus orchromatin mass. The reprogrammed cell formed from this step isadministered to a mammal in need of that cell type. In one preferredembodiment, the permeabilized cell is incubated with an interphasereprogramming media. Preferably, the nucleus in the permeabilized cellremains membrane-bounded, and the chromosomes in the nucleus do notcondense during incubation with the interphase reprogramming media. Inanother preferred embodiment, a chromatin mass is formed from incubationof the permeabilized cell in a mitotic reprogramming media. In yetanother preferred embodiment, the reprogrammed cell is incubated underconditions that allow the membrane of the reprogrammed cell to resealprior to being administered to the mammal. Preferably, the permeabilizedcell is from the mammal in need of that cell type. In another preferredembodiment, the permeabilized cell is formed by incubating an intactcell with a detergent, such as digitonin, or a bacterial toxin, such asStreptolysin O. Examples of diseases, disorders, or conditions that maybe treated or prevented include neurological, endocrine, structural,skeletal, vascular, urinary, digestive, integumentary, blood, immune,auto-immune, inflammatory, endocrine, kidney, bladder, cardiovascular,cancer, circulatory, digestive, hematopoeitic, and muscular diseases,disorders, and conditions. In addition, reprogrammed cells may be usedfor reconstructive applications, such as for repairing or replacingtissues or organs.

The invention also provides methods for measuring an endogenous activity(e.g., an endogenous enzymatic activity) or an endogenous protein in acell, nucleus, chromatin mass, cell lysate, or in vitro sample. In onesuch aspect, the method involves contacting a solid support with a testsample from a cell, nucleus, chromatin mass, cell lysate, or in vitrosample, and with a reference sample. The test sample has an endogenousactivity of interest that is naturally found in the test sample (e.g.,luciferase activity or phosphatase activity, e.g., alkaline phosphataseactivity), and the test sample has a known protein concentration or isderived from a known number of cells. The reference sample has a knownlevel of the activity of interest (e.g., luciferase activity orphosphatase activity) or a known amount of naturally-occurring orrecombinant protein having the activity. The level of luciferase orphosphatase activity in the test sample is measured and compared to thelevel of luciferase or phosphatase activity in the reference sample,thereby determining the level of luciferase or phosphatase activity inthe cell, nucleus, chromatin mass, cell lysate, or in vitro sample. Inone preferred embodiment, the luciferase or phosphatase activity isperformed by a naturally-occurring protein encoded by an endogenousnucleic acid under the control of an endogenous promoter. This methodmay also be used to measure any other endogenous activity of interest.In another preferred embodiment, the activity is specific for one celltype or specific for a family of related cell types. In variousembodiments, the activity of interest is the chemical alteration of oneor more substrates to form a product. Preferably, either one of thesubstrates or one of the products is detectable. Detectable labels arewell known in the art and include, without limitation, radioactivelabels (e.g., isotopes such as ³²p or ³⁵S) and nonradioactive labels(e.g., chemiluminescent labels or fluorescent labels, e.g.,fluorescein).

In a related aspect, the invention provides a method for measuring thelevel of an endogenous protein in a cell, nucleus, chromatin mass, celllysate, or in vitro sample. This method involves contacting a solidsupport with a test sample from a cell, nucleus, chromatin mass, celllysate, or in vitro sample, and with a reference sample. The test samplehas an endogenous, detectable protein of interest (e.g., luciferase,alkaline phosphatase, or Oct4) that is naturally found in the testsample, and the test sample has a known protein concentration or isderived from a known number of cells. The reference sample has a knownamount of the protein of interest, e.g., naturally-occurring orrecombinant luciferase, alkaline phosphatase, or Oct4 protein. Thesignal from the luciferase, alkaline phosphatase, or Oct4 protein in thetest sample is measured and compared to the signal from thecorresponding protein in the reference sample, thereby determining theamount of luciferase, alkaline phosphatase, or Oct4 protein in the cell,nucleus, chromatin mass, cell lysate, or in vitro sample. This methodmay also be used to measure the level of any other endogenous protein ofinterest. In one preferred embodiment, the protein of interest isencoded by an endogenous nucleic acid under the control of an endogenouspromoter. In a preferred embodiment, the protein of interest is specificfor one cell type or specific for a family of related cell types. Invarious embodiments, the protein of interest has a detectable label orbinds another molecule (e.g., an antibody) with a detectable label.Exemplary detectable labels include radioactive labels (e.g., isotopessuch as ³²P or ³⁵S) and nonradioactive labels (e.g., chemiluminescentlabels or fluorescent labels, e.g., fluorescein).

In preferred embodiments for the above methods of measuring the level ofan activity or protein of interest, the solid support is contacted withmultiple reference samples, each with a different level of activity or adifferent amount of the protein of interest. According to thisembodiment, a standard curve may be generated from the reference samplesand used to determine the level of activity or the amount of the proteinof interest in the test sample. In various embodiments, the cell is astem cell such as an embryonic stem cell or an adult stem cell frombrain, blood, bone marrow, pancreas, liver, skin, or any other organ ortissue. In other embodiments, the cell has been exposed to an extractfrom a stem cell. In yet other embodiments, the test sample is a from anuclear or cytoplasmic cell extract. Useful solid supports include anyrigid or semi-rigid surface that may be contacted with the sample. Thesupport can be any porous or non-porous water insoluble material,including, without limitation, membranes, filters, chips, slides,fibers, beads, gels, tubing, strips, plates, rods, polymers, particles,microparticles, capillaries, and plastic surfaces. If desired, thesupport can have a variety of surface forms, such as wells, trenches,pins, channels and pores, to which the samples are contacted.

In preferred embodiments of various aspects of the invention, at least1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 300, or more mRNA orprotein molecules are expressed in the reprogrammed cell that are notexpressed in the donor or permeabilized cell. In another preferredembodiment, the number of mRNA or protein molecules that are expressedin the reprogrammed cell, but not expressed in the donor orpermeabilized cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50,50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300,inclusive. Preferably, at least 1, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 300, or more mRNA or protein molecules are expressed in the donoror permeabilized cell that are not expressed in the reprogrammed cell.In yet another preferred embodiment, the number of mRNA or proteinmolecules that are expressed in the donor or permeabilized cell, but notexpressed in the reprogrammed cell, is between 1 and 5, 5 and 10, 10 and25, 25 and 50, 50 and 75, 75 and 100, 100 and 150, 150 and 200, or 200and 300, inclusive. Preferably, the mRNA or protein molecules arespecific for the cell type of the donor, permeabilized, or reprogrammedcell, such that the molecules are only expressed in cells of thatparticular cell type. In still another preferred embodiment, these mRNAor protein molecules are expressed in both the donor cell (i.e., thedonor or permeabilized starting cell) and the reprogrammed cell, but theexpression levels in these cells differ by at least 2, 5, 10, or20-fold, as measured using standard assays (see, for example, Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, 2000). In other embodiments, the expression of one or morecytoskeleton proteins such as integrins is decreased by at least atleast 2, 5, 10, or 20-fold compared to the donor fibroblast cell. In yetother embodiments, the reprogrammed cell expresses a neurofilamentprotein, T-cell receptor protein, IL-2, IL-2 receptor, insulin, or Oct4at a level that is at least 2, 5, 10, or 20-fold greater that thecorresponding level in the donor or permeabilized cell.

In other preferred embodiments, the size of the donor or permeabilizedcell differs from that of the reprogrammed cell by at least 10, 20, 30,50, 75, or 100%, as measured using standard methods. In anotherpreferred embodiment, the volume of cytoplasm in the donor orpermeabilized cell differs from that in the reprogrammed cell by atleast 10, 20, 30, 50, 75, or 100%, based on standard methods. In yetanother preferred embodiment, the reprogrammed cell has gained or lostan activity relative to the donor or permeabilized cell, such assecretion of a particular hormone, extracellular matrix component, orantibody. In another embodiment, the reprogrammed cell has gained theability to produce and secrete an interleukin such as IL-2, gained theability to form a neurofilament, neurite, or axon, or gained the abilityto form an embryoid body. In another embodiment, cell has gained theability to express a T-cell receptor or IL-2 receptor or to produceinsulin. Preferably, the β-chain of the IL-2 receptor is expressedconstitutively, and the α-chain is expressed upon activation (e.g., bystimulation with an anti-CD3 antibody or by presentation of an antigen).In other embodiments, a reprogrammed cell such as a stem cell orfibroblast has gained the ability to contract, resembling a contractingmuscle cell or beating heart cell.

In still other preferred embodiments, the reprogramming media is aninterphase reprogramming media, such as an extract formed from cellssynchronized in one or more of the following phases of the cell cycle:G_(o), G₁, S, or G₂ phase. In another preferred embodiment, thereprogramming media is an extract formed from cells synchronized inmitosis or from unsynchronized cells. Preferably, the reprogrammingmedia is an extract from the cell type one wishes the donor orpermeabilized cell to become, or the reprogramming media is a solutioncontaining factors specific for the cell type one wishes the donor orpermeabilized cell to become. Examples of cells that may be used togenerate extracts to reprogram cells into stem cells include embryonicstem cells and adult stem cells from brain, blood, bone marrow,pancreas, liver, skin, or any other organ or tissue. Preferably, thedonor or permeabilized cell is an interphase or mitotic somatic cell. Inanother preferred embodiment, the reprogramming media is modified by theenrichment or depletion of a factor, such as a DNA methyltransferase,histone deacetylase, histone, nuclear lamin, transcription factor,activator, repressor, growth factor, hormone, or cytokine. Thereprogramming media may or may not contain exogenous nucleotides. Inother preferred embodiments, a chromatin mass in a reprogramming mediaor formed in a permeabilized cell is contacted with a vector having anucleic acid encoding a gene of interest under conditions that allowhomologous recombination between the nucleic acid in the vector and thecorresponding nucleic acid in the genome of the chromatin mass,resulting in the alteration of the genome of the chromatin mass. Due tothe lack of an intact plasma membrane and the lack of a nuclearmembrane, a chromatin mass in a permeabilized cell may be easier togenetically modify than a naturally-occurring cell. Preferably, thechromatin mass or nucleus is purified from the reprogramming media priorto insertion into the recipient cell or cytoplast, or the reprogrammedcell is purified prior to administration into the mammal. Preferably,the donor or permeabilized cell is haploid (DNA content of n), diploid(2n), or tetraploid (4n), and the recipient cell is hypodiploid (DNAcontent of less than 2n), haploid, or enucleated.

Preferred donor cells, permeabilized cells, recipient cells,reprogrammed cells, and sources of cytoplasts include differentiatedcells, such as epithelial cells, neural cells, epidermal cells,keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-cells,T-cells, erythrocytes, macrophages, monocytes, fibroblasts, and musclecells; and undifferentiated cells, such as embryonic or adult stemcells. In another preferred embodiment, the donor or permeabilized cellis a differentiated cell, and the reprogrammed cell is a differentiatedcell of another cell type. In yet another preferred embodiment, thedonor or permeabilized cell is an undifferentiated cell, and thereprogrammed cell is a differentiated cell. In still another preferredembodiment, the donor or permeabilized cell is a differentiated cell,and the reprogrammed cell is an undifferentiated cell. If desired, anundifferentiated reprogrammed cell may be induced to differentiate intoa desired cell type in vitro using standard methods, such as by exposureto certain growth factors, hormones, interleukins, cytokines, or othercells. In another preferred embodiment, the undifferentiatedreprogrammed cell differentiates into a desired cell type in vivo afteradministration to a mammal. In yet another preferred embodiment, thedonor or permeabilized cell is a B-cell, Jurkat cell, endothelial cell,epithelial cell, or fibroblast, and the reprogrammed cell is a T-cell.It is also contemplated that the nucleus or chromatin mass may beinserted into a recipient cell or cytoplast of the desired cell type orof the same cell type as the donor or permeabilized cell. In stillanother preferred embodiment, the donor cell, permeabilized cell,recipient cell, or recipient cytoplast is from a human or non-humanmammal. In yet another preferred embodiment, the donor nucleus orchromatin mass is from a transgenic cell or mammal or contains amutation not found in the donor cell or not found in anaturally-occurring cell. The donor or permeabilized cell can benon-immortilized or naturally, spontaneously, or geneticallyimmortilized. The donor cell, permeabilized cell, recipient cell, orcytoplast can be from a source of any age, such as an embryo, fetus,youth, or adult mammal. Cells from younger sources may have acquiredfewer spontaneous mutations and may have a longer life-span in vitro orafter transplantation in vivo.

Preferably, a disease-causing mutation in a regulatory region, promoter,untranslated region, or coding region of a gene in a donor nucleus orchromatin mass is modified to replace the mutant sequence with asequence that is not associated with the disease. Alternatively, anucleic acid is inserted into the donor nucleus or chromatin mass thatincludes a promoter operably-linked to a sequence of the gene that doesnot contain a mutation associated with a disease. Preferably, thesequence of the gene is substantially identical to that of anaturally-occurring gene that does not contain a polymorphism ormutation associated with a disease. Examples of mutations that may berescued using these methods include mutations in the cystic fibrosisgene; mutations associated with Dunningan's disease such as the R482W,R482Q, and R584H mutations in the lamin A gene; and mutations associatedwith the autosomal-dominant form of Emery Deyfuss muscular dystrophysuch as the R249Q, R453W, and Q6STOP mutations in the lamin A gene. Inthe Q6STOP mutation, the codon for Gln6 is mutated to a stop codon.

Preferred transgenic donor nuclei, chromosomes, or chromatin massesencode a heterologous MHC Class 1 protein having an amino acid sequencesubstantially identical to the sequence of an MHC Class 1 protein foundin the mammal to whom the reprogrammed cells will be administered fortherapeutic applications. Alternatively, the donor nuclei or chromatinmasses may encode a heterologous MHC Class 1 protein having an aminoacid sequence substantially identical to the sequence of an MHC Class 1protein found in another mammal of the same genus or species as therecipient mammal. Reprogrammed cells that express such MHC proteins areless likely to elicit an adverse immune response when administered tothe mammal. Other preferred donor nuclei or chromatin masses aremodified to express a heterologous protein that inhibits the complementpathway of the recipient mammal, such as the human complement inhibitorCD59 or the human complement regulator decay accelerating factor (h-DAF)(see, for example, Ramirez et al., Transplantation 15:989-998, 2000;Costa et al., Xenotransplantation 6:6-16, 1999). In yet anotherpreferred embodiment, the donor nucleus or chromatin mass has a mutationthat reduces or eliminates the expression or activity of agalactosyltransferase, such as alpha(1,3)-galactosyltransferase (Tearleet al., Transplantation 61:13-19, 1996; Sandrin, Immunol. Rev.141:169-190, 1994; Costa et al., Xenotransplantation 6:6-16, 1999). Thisenzyme modifies cell surface molecules with a carbohydrate that elicitsan adverse immune response when cells expressing this galactosealpha(1,3)-galactose epitope are administered to humans. Thus,reprogrammed cells that have a lower level of expression of this epitopemay have a lower incidence of rejection by the recipient mammal.

With respect to the therapeutic methods of the invention, it is notintended that the administration of reprogrammed cells to a mammal belimited to a particular mode of administration, dosage, or frequency ofdosing; the present invention contemplates all modes of administration,including intramuscular, intravenous, intraarticular, intralesional,subcutaneous, or any other route sufficient to provide a dose adequateto prevent or treat a disease. Preferably, the cells are administered tothe mammal from which the donor or permeabilized cell is obtained.Alternatively, the donor or permeabilized cell may be obtained from adifferent donor mammal of the same or a different genus or species asthe recipient mammal. Examples of preferred donor mammals includehumans, cows, sheep, big-horn sheep, goats, buffalos, antelopes, oxen,horses, donkeys, mule, deer, elk, caribou, water buffalo, camels, llama,alpaca, rabbits, pigs, mice, rats, guinea pigs, hamsters, dogs, cats,and primates such as monkeys. The cells may be administered to themammal in a single dose or multiple doses. When multiple doses areadministered, the doses may be separated from one another by, forexample, one week, one month, one year, or ten years. One or more growthfactors, hormones, interleukins, cytokines, or other cells may also beadministered before, during, or after administration of the cells tofurther bias them towards a particular cell type. Additionally, one ormore immunosuppressive agents, such as cyclosporin, may be administeredto inhibit rejection of the transplanted cells. It is to be understoodthat, for any particular subject, specific dosage regimes should beadjusted over time according to the individual need and the professionaljudgment of the person administering or supervising the administrationof the compositions.

As used herein, by “chromatin mass” is meant more than one chromosomenot enclosed by a membrane. Preferably, the chromatin mass contains allof the chromosomes of a cell. A chromatin mass containing condensedchromosomes may be formed by exposure of a nucleus to a mitoticreprogramming media (e.g., a mitotic extract), or a chromatin mass maybe isolated from mitotic cells as described herein. Alternatively, achromatin mass containing decondensed or partially condensed chromosomesmay be generated by exposure of a nucleus to one of the following, asdescribed herein: a mitotic reprogramming media (e.g., a mitoticextract) in the presence of an anti-NuMA antibody, a detergent and saltsolution, or a protein kinase solution.

A chromatin mass may be formed naturally or artificially induced. Anexemplary naturally-occurring chromatin mass includes a set of metaphasechromosomes, which are partially or maximally condensed chromosomes thatare not surrounded by a membrane and that are found in, or isolatedfrom, a mitotic cell. Preferably, the metaphase chromosomes are discretechromosomes that are not physically touching each other. Exemplaryartificially induced chromatin masses are formed from exposure to areprogramming media, such as a solution containing factors that promotechromosome condensation, a mitotic extract, a detergent and saltsolution, or a protein kinase solution. Artificially induced chromatinmasses may contain discrete chromosomes that are not physically touchingeach other or may contain two or more chromosomes that are in physicalcontact.

If desired, the level of chromosome condensation may be determined usingstandard methods by measuring the intensity of staining with the DNAstain, DAPI. As chromosomes condense, this staining intensity increases.Thus, the staining intensity of the chromosomes may be compared to thestaining intensity for decondensed chromosomes in interphase (designated0% condensed) and maximally condensed chromosomes in mitosis (designated100% condensed). Based on this comparison, the percent of maximalcondensation may be determined. Preferred condensed chromatin masses areat least 50, 60, 70, 80, 90, or 100% condensed. Preferred decondensed orpartially condensed chromatin masses are less than 50, 40, 30, 20, or10% condensed.

By “nucleus” is meant a membrane-bounded organelle containing most orall of the DNA of a cell. The DNA is packaged into chromosomes in adecondensed form. Preferably, the membrane encapsulating the DNAincludes one or two lipid bilayers or has nucleoporins.

By “donor cell” is meant a cell from which a nucleus or chromatin massis derived.

By “cytoplast” is meant a membrane-enclosed cytoplasm. Preferably, thecytoplast does not contain a nucleus, chromatin mass, or chromosome.Cytoplasts may be formed using standard procedures. For example,cytoplasts may be derived from nucleated or enucleated cells.Alternatively, cytoplasts may be generated using methods that do notrequire an intact cell to be used as the source of the cytoplasm or asthe source of the membrane. In one such method, cytoplasts are producedby the formation of a membrane in the presence of cytoplasm underconditions that allow encapsulation of the cytoplasm by the membrane.

By “permeabilization” is meant the formation of pores in the plasmamembrane or the partial or complete removal of the plasma membrane.

By “reprogramming media” is meant a solution that allows the removal ofa factor from a nucleus, chromatin mass, or chromosome or the additionof a factor from the solution to the nucleus, chromatin mass, orchromosome. Preferably, the addition or removal of a factor increases ordecreases the level of expression of an mRNA or protein in the donorcell, chromatin mass, or nucleus or in a cell containing thereprogrammed chromatin mass or nucleus. In another embodiment,incubating a permeabilized cell, chromatin mass, or nucleus in thereprogramming media alters a phenotype of the permeabilized cell or acell containing the reprogrammed chromatin mass or nucleus relative tothe phenotype of the donor cell. In yet another embodiment, incubating apermeabilized cell, chromatin mass, or nucleus in the reprogrammingmedia causes the permeabilized cell or a cell containing thereprogrammed chromatin mass or nucleus to gain or loss an activityrelative to the donor cell.

Exemplary reprogramming medias include solutions, such as buffers, thatdo not contain biological molecules such as proteins or nucleic acids.Such solutions are useful for the removal of one or more factors from anucleus, chromatin mass, or chromosome. Other preferred reprogrammingmedias are extracts, such as cellular extracts from cell nuclei, cellcytoplasm, or a combination thereof. Yet other reprogramming medias aresolutions or extracts to which one or more naturally-occurring orrecombinant factors (e.g., nucleic acids or proteins such as DNAmethyltransferases, histone deacetylases, histones, nuclear lamins,transcription factors, activators, repressors, growth factors, hormones,or cytokines) have been added, or extracts from which one or morefactors have been removed. Still other reprogramming medias includedetergent and salt solutions and protein kinase solutions. In someembodiments, the reprogramming media contains an anti-NuMA antibody. By“interphase reprogramming media” is meant a media (e.g., an interphasecell extract) that induces chromatin decondensation and nuclear envelopeformation. By “mitotic reprogramming media” is meant a media (e.g., amitotic cell extract) that induces chromatin condensation and nuclearenvelope breakdown. If desired, multiple reprogramming media may be usedsimultaneously or sequentially to reprogram a donor cell, nucleus, orchromatin mass.

By “addition of a factor” is meant the binding of a factor to chromatin,a chromosome, or a component of the nuclear envelope, such as thenuclear membrane or nuclear matrix. Alternatively, the factor isimported into the nucleus so that it is bounded or encapsulated by thenuclear envelope. Preferably, the amount of factor that is bound to achromosome or located in the nucleus increases by at least 25, 50, 75,100, 200, or 500%.

By “removal of factor” is meant the dissociation of a factor fromchromatin, a chromosome, or a component of the nuclear envelope, such asthe nuclear membrane or nuclear matrix. Alternatively, the factor isexported out of the nucleus so that it is no longer bounded orencapsulated by the nuclear envelope. Preferably, the amount of factorthat is bound to a chromosome or located in the nucleus decreases by atleast 25, 50, 75, 100, 200, or 500%.

By “enrichment or depletion of a factor” is meant the addition orremoval of a naturally-occurring or recombinant factor by at least 20,40, 60, 80, or 100% of the amount of the factor originally present inthe reprogramming media. Alternatively, a naturally-occurring orrecombinant factor that is not naturally present in the reprogrammingmedia may be added. Preferred factors include proteins such as DNAmethyltransferases, histone deacetylases, histones, nuclear lamins,transcription factors, activators, repressors, growth factors,cytokines, and hormones; membrane vesicles; and organelles. In onepreferred embodiment, the factor is purified prior to being added to thereprogramming media, as described below. Alternatively, one of thepurification methods described below may be used to remove an undesiredfactor from the reprogramming media.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor is substantially pure when it is atleast 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the factor is at least 75%, more preferably, atleast 90%, and most preferably, at least 99%, by weight, pure. Asubstantially pure factor may be obtained by chemical synthesis,separation of the factor from natural sources, or production of thefactor in a recombinant host cell that does not naturally produce thefactor. Proteins, vesicles, chromosomes, nuclei, and other organellesmay be purified by one skilled in the art using standard techniques suchas those described by Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, New York, 2000). The factor is preferably atleast 2, 5, or 10 times as pure as the starting material, as measuredusing polyacrylamide gel electrophoresis, column chromatography, opticaldensity, HPLC analysis, or western analysis (Ausubel et al., supra).Preferred methods of purification include immunoprecipitation, columnchromatography such as immunoaffinity chromatography, magnetic beadimmunoaffinity purification, and panning with a plate-bound antibody.

By “mRNA or protein specific for one cell type” is meant an mRNA orprotein that is expressed in one cell type at a level that is at least10, 20, 50, 75, or 100 fold greater than the expression level in allother cell types. Preferably, the mRNA or protein is only expressed inone cell type.

By “mutation” is meant an alteration in a naturally-occurring orreference nucleic acid sequence, such as an insertion, deletion,frameshift mutation, silent mutation, nonsense mutation, or missensemutation. Preferably, the amino acid sequence encoded by the nucleicacid sequence has at least one amino acid alteration from anaturally-occurring sequence. Examples of recombinant DNA techniques foraltering the genomic sequence of a cell, embryo, fetus, or mammalinclude inserting a DNA sequence from another organism (e.g., a human)into the genome, deleting one or more DNA sequences, and introducing oneor more base mutations (e.g., site-directed or random mutations) into atarget DNA sequence. Examples of methods for producing thesemodifications include retroviral insertion, artificial chromosometechniques, gene insertion, random insertion with tissue specificpromoters, homologous recombination, gene targeting, transposableelements, and any other method for introducing foreign DNA. All of thesetechniques are well known to those skilled in the art of molecularbiology (see, for example, Ausubel et al., supra). Chromatin masses,chromosomes, and nuclei from transgenic cells, tissues, organs, ormammals containing modified DNA may be used in the methods of theinvention.

By “substantially identical” is meant having a sequence that is at least60, 70, 80, 90, or 100% identical to that of another sequence or to anaturally-occurring sequence. Sequence identity is typically measuredusing sequence analysis software with the default parameters specifiedtherein (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). This software program matchessimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

By “immortilized” is meant capable of undergoing at least 25, 50, 75,90, or 95% more cell divisions than a naturally-occurring control cellof the same cell type, genus, and species as the immortalized cell orthan the donor cell from which the immortalized cell was derived.Preferably, an immortalized cell is capable of undergoing at least 2, 5,10, or 20-fold more cell divisions than the control cell. Morepreferably, the immortalized cell is capable of undergoing an unlimitednumber of cell divisions. Examples of immortalized cells include cellsthat naturally acquire a mutation in vivo or in vitro that alters theirnormal growth-regulating process. Other preferred immortalized cellsinclude hybridoma cells which are generated using standard techniquesfor fusion of a myeloma with a B-cell (Mocikat, J. Immunol. Methods225:185-189, 1999; Jonak et al., Hum. Antibodies Hybridomas 3:177-185,1992; Srikumaran et al., Science 220:522, 1983). Still other preferredimmortalized cells include cells that have been genetically modified toexpress an oncogene, such as ras, myc, abl, bcl2, or neu, or that havebeen infected with a transforming DNA or RNA virus, such as Epstein Barrvirus or SV40 virus (Kumar et al., Immunol. Lett. 65:153-159, 1999;Knight et al., Proc. Nat. Acad. Sci. USA 85:3130-3134, 1988; Shammah etal., J. Immunol. Methods 160-19-25, 1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98-104, 1994; Kataoka et al., Differentiation62:201-211, 1997; Chatelut et al., Scand. J. Immunol. 48:659-666, 1998).Cells can also be genetically modified to express the telomerase gene(Roques et al., Cancer Res. 61:8405-8507, 2001).

By “non-immortilized” is meant not immortalized as described above.

The present invention provides a number of advantages related to thealteration of cell fate. For example, these methods may be generallyapplied to produce cells of any desired cell type. Because these methodsinvolve incubating a nucleus, a chromatin mass, or a permeabilized cellin a reprogramming media (e.g., a cell extract) to allow reprogramming,the efficiency of reprogramming may be enhanced by adding factors to thereprogramming media that facilitate reprogramming or by removing factorsthat inhibit reprogramming. These reprogrammed cells may be transplantedinto mammals for the treatment or prevention of conditions involvingdamage or deficiency of a particular cell type. If desired, thereprogrammed cells may be manipulated using standard molecular biologytechniques to correct a disease-causing mutation before administeringthe cells to a recipient mammal.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains drawings executed in color (FIGS. 13, 14A,14B, 14C, 15, and 17A). Copies of this patent or patent application withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is an illustration of a gel showing the amount of IL-2 mRNAsynthesized by human T-cells stimulated with anti-CD3 antibodies for theindicated lengths of time. Lanes “30c,” “60c,” and “120c” refer tomock-stimulated T-cells.

FIG. 2 is an illustration of nuclei purified from resting T-cells.

FIG. 3A is the immunofluorescence analysis of nuclear uptake and bindingof the T-cell specific transcription factor NFAT in cell-freereprogramming extracts. For this assay, nuclei purified from restingT-cells (“Input” nuclei) were incubated with reprogramming extract fromstimulated T-cells for 30 minutes. FIG. 3B in an illustration of animmunoblot showing the nuclear uptake of NFAT, c-Jun/AP1, NFκB, Oct1,and MAP kinase (Erk1 and Erk2). Input nuclei (“Input”) and nucleiincubated in either a stimulated extract (“SE”), a control extractprepared from unstimulated T-cells (denoted “USE” for unstimulatedextract), or a stimulated extract containing a monoclonal antibodyagainst nucleoporins (“SE+mAb414”) were analyzed. FIG. 3C is a bar graphshowing the percentage of transcription factors bound to DNA, reportedas the mean±the standard deviation.

FIG. 4A is an illustration of an immunoblot showing the DNA-binding ofthe chromatin remodeling SWI/SNF complex in T-cell nuclei exposed to abuffer control, an unstimulated extract, or a stimulated extract for 30minutes and sedimented through sucrose. FIG. 4B is a graph showing thepercentage of DNA bound and unbound SWI/SNF complex in nuclei incubatedin extract for the indicated lengths of time, as assessed using anuclear retention assay. The SWI/SNF complex was visualized on Westernblots using anti-BRG1 antibodies. FIG. 4C is an illustration of animmunoblot showing the nuclear retention of the SWI/SNF complex instimulated T-cells in vivo. For this assay, T-cells were stimulated withanti-CD3 antibodies for 30 minutes, and the soluble and DNA boundfractions of the SWI/SNF complex were assessed as described for FIG. 4A.FIG. 4D is an illustration of an immunoblot showing hyperacetylation ofthe IL-2 gene of T-cell nuclei in vivo and in vitro. An MNAse-solublechromatin fraction was prepared from resting and anti-CD3-stimulatedT-cells. The immunoblot shows the binding of an IL-2 probe and a controlβ-actin probe to DNA from the immunoprecipitate of the chromatinfraction with an anti-acetylated histone H4 (H4ac) probe (“bound”) andthe binding to DNA in the supernatant (“unbound”) fractions.

FIG. 5A is a gel showing the synthesis of IL-2 mRNA in cell-freeextracts. Resting T-cell nuclei (“Input nuclei”) were incubated for 30minutes at 30° C. in an unstimulated extract or a stimulated extract. Ascontrols, nuclei were incubated in a stimulated extract containingeither 100 μg/ml RNAse A, 100 μg/ml DNAse I, mAb414 antibodies, or thelectin WGA. FIG. 5B is a gel showing that IL-2 mRNA production in vitrois dependent on PolII transcription. Nuclei were exposed for 30 minutesto a stimulated extract containing increasing concentrations of the RNAPolII inhibitor actinomycin D (0, 5, 10, 50, 100 and 500 nM), and IL-2mRNA synthesis was analyzed by reverse transcription-polymerase chainreaction (RT-PCR). FIG. 5C is an illustration of a gel showing that IL-2mRNA production was induced in nuclei exposed to a T-cell extracttreated with anti-CD3 antibodies but not induced by exposure to extractstreated with anti-CD3 antibodies from the B-cell line Reh, human 293Tfibroblasts, or HeLa cells. The arrows in the illustration point to the467 base pair IL-2 RT-PCR product.

FIG. 6A is the immunofluorescence analysis showing nuclear uptake andbinding of the T-cell specific transcription factor NFAT by nuclei fromthe B-cell line Reh, 293T fibroblasts, or HeLa cells that have beenincubated with the stimulated T-cell extract, as described for FIG. 3A.FIG. 6B in an illustration of an immunoblot showing the nuclear uptakeof NFAT, c-Jun/AP1, and NFκKB by these nuclei, as described for FIG. 3B.

FIG. 7 is a picture of a gel showing RT-PCR analysis of IL-2 mRNAsynthesis in human peripheral blood T-cells stimulated with anti-CD3antibodies. At indicated time points, cells were lysed, and total RNAwas isolated for RT-PCR analysis using IL-2-specific primers. Mock(H₂O)-stimulated cells were analyzed at 30, 60, and 120 minute timepoints (“30c,” “60c,” “120c”)

FIGS. 8A-8D are pictures and graphs that illustrate nuclear uptake andchromatin binding of transcriptional activators of the IL-2 gene instimulated T-cell extract. FIG. 8A is a picture of nuclei purified fromquiescent T-cells (“Input nuclei”) and incubated in stimulated extractfor 30 minutes. Nuclear integrity was assessed by phase contrastmicroscopy and membrane labeling with 10 μg/ml of the lipophilic dyeDiOC₆ (bar, 10 μm). FIG. 8B is a picture of an immunoblot showing therelative levels of NFAT, AP-1, NFκB, Oct-1, Erk, and an exogenousBSA-NLS conjugate in input resting T-cell nuclei (“Input nuclei”), inputunstimulated extract, and input stimulated extract. Nuclear uptake ofthese factors was examined in nuclei exposed to stimulated extract,unstimulated extract, or stimulated extract containing the nuclear poreblocking antibody mAb414. Blots were also probed using anti-histone H4antibodies as a gel loading control. FIG. 8C is a graph of theimmunoblotting analysis of intranuclear anchoring of importedtranscription factors in nuclei exposed to unstimulated extract orstimulated extract. Intranuclear anchoring was assessed using a nuclearretention assay and immunoblotting analysis of Triton X-100 insoluble(“bound”) fractions. The percentage (mean±SD) of bound factors wasdetermined by densitometric analysis of duplicate blots. FIG. 8D is apicture of immunoblotting analysis of nuclear matrix (“Mtx”) andchromatin (“Chr”) fractions prepared from nuclei exposed to stimulatedextract. NuMA and RNA Pol IIo were used as markers of the nuclear matrixand chromatin, respectively. Anti-NuMA antibodies and anti-Pol IIo mAbCC3 were gifts from D. Compton (Dartmouth Medical School, Hanover, N.H.,USA) and M. Vincent (Université Laval, Quebec, Canada), respectively.

FIGS. 9A-9E illustrate chromatin remodeling in resting T-cell nucleiexposed to a stimulated T-cell extract. FIG. 9A is a gel showing theanchoring of the chromatin remodeling complex SWI/SNF in nuclei exposedto stimulated extract. Purified quiescent T-cell nuclei (“Input nuclei”)were incubated in cell lysis buffer (“Buffer”), unstimulated extract, orstimulated extract for 30 minutes, and sedimented through sucrose. Freeand bound SWI/SNF fractions were assessed using the nuclear retentionassay and immunoblotting with anti-BRG1 antibodies. FIG. 9B is a graphof the percentage of bound SWI/SNF, as determined using the nuclearretention assay during incubation of nuclei in stimulated extract orunstimulated extract. FIG. 9C is a gel showing nuclear retention ofSWI/SNF in cultured stimulated T-cells. T-cells were stimulated withanti-CD3 antibodies and after 30 minutes, intranuclear bound and freefractions of SWI/SNF were assessed as in FIG. 9A. FIG. 9D is a graph ofthe ATPase activity of the SWI/SNF complex. After incubation of restingT-cell nuclei in stimulated extract or unstimulated extract for 30minutes, SWI/SNF was immunoprecipitated from nuclear lysates usinganti-BRG1 antibodies and hydrolysis of 1 nM exogenous ATP (“ATP+”) bythe immune precipitate (“BRG1-IP”) was determined in a luminometricassay. Control precipitations were carried out using pre-immune IgGs(“Pre-I IgG”). Top rows show an anti-BRG1 immunoblot of the BRG1-IPs.Where indicated, BRG1-IP was treated with 100 μg/ml DNAse I for 15minutes prior to the assay (“DNAse+”). Relative ATPase activities areexpressed as mean (±SD) relative light units (RLU) in the assaysubtracted to the RLU of the control pre-immune precipitate (RLU=2,700,set to zero). FIG. 9E is a gel showing the hyperacetylation of the IL-2promoter in T-cell nuclei in culture and in vitro. Microccocalnuclease-soluble chromatin (“Input”) was prepared from unstimulated(“Unstim.”) and anti-CD3-stimulated (“Stim.”) T-cells. AcH4 wasimmunoprecipitated, and DNA isolated from anti-acH4 precipitates(“Bound”) and supernatant (“Unbound”) fractions. DNA was dot-blotted andhybridized with an IL-2 promoter-specific probe and a control β-actinprobe.

FIGS. 10A-10D are gels showing the transcription of IL-2 mRNA by T-celland non-T-cell nuclei in cell-free extracts. FIG. 10A is a gel showingtranscription by resting T-cell nuclei (“Input nuclei”) incubated instimulated extract (“Nuclei-SE”) or unstimulated extract (“Nuclei-USE”).As controls, nuclei were incubated in stimulated extract containingeither 100 μg/ml RNAse A, 100 μg/ml DNAse I, mAb414, or wheat germagglutinin (“WGA”). At the end of the incubation, total RNA was isolatedfrom the reaction mix, and 15 ng was used as the template for RT-PCRusing IL-2-specific primers. Input stimulated extract and stimulatedextract containing 1.2 μg total RNA isolated from IL-2 producing T-cells(“Pos. control SE”) were analyzed as controls. FIG. 10B is a picture ofa gel showing that IL-2 mRNA synthesis in vitro is RNA Pol II-dependent.Resting T-cell nuclei were exposed to stimulated extract containing 0,5, 10, 50, 100 or 500 nM of the RNA Pol II inhibitor, actinomycin D.IL-2 mRNA synthesis was analyzed by RT-PCR at the end of incubation.FIG. 10C is a gel showing transcription by nuclei from primary HUVECcells, NT2 cells, and Hela cells that were reprogrammed for 2 hours instimulated extract or unstimulated extract. As controls, nuclei wereincubated in stimulated extract containing either 100 μg/ml RNAse A,mAb414, or 50 nM actinomycin D (“ActD”). Total RNA was isolated, andIL-2 RNA synthesis was examined by RT-PCR. FIG. 10D is a picture of agel showing transcription by resting T-cell nuclei that were incubatedfor two hours in stimulated extract, or in extracts from 293T, HeLa, orBjab cells, all prepared after treating each cell type with anti-CD3 andcross-linking antibodies to mimic T-cell stimulation. IL-2 mRNAsynthesis in each extract was analyzed by RT-PCR.

FIGS. 11A-11C demonstrate the nuclear import and chromatin binding oftranscriptional activators of the IL-2 gene in 293T fibroblast nucleiexposed to stimulated T-cell extract. FIG. 11A is a picture of nucleipurified from 293T fibroblasts (0 min) and incubated in stimulatedT-cell extract. Uptake of NFAT was examined by immunofluorescence (bar,10 μm). FIG. 11B is a picture of immunoblotting analysis showing nuclearuptake of NFAT, AP-1, NFκB, Oct-1, and BSA-NLS in input 293T nuclei and293T nuclei exposed to either stimulated T-cell extract, unstimulatedT-cell extract, or stimulated T-cell extract and mAb414. Anti-histone H4antibodies were used as a loading control. FIG. 11C is a picture of animmunoblot showing nuclear matrix (“Mtx”) and chromatin (“Chr”)fractions prepared from 293T nuclei treated with stimulated T-cellextract.

FIGS. 12A-2E demonstrate chromatin remodeling and activation of the IL-2gene in stimulated T-cell extract. FIG. 12A is a gel showingintranuclear anchoring of the human SWI/SNF complex. In the left sectionof the immunoblot, nuclei were isolated from resting (“−”) oranti-CD3-stimulated (“α-CD3”) T-cells, and intranuclear free and boundSWI/SNF assessed by immunoblotting of detergent-soluble and insolublenuclear fractions using anti-BRG1 antibodies. In the right section ofthe immunoblot, free and bound SWI/SNF fractions were visualized in 293Tnuclei incubated in stimulated T-cell extract and sedimented throughsucrose. FIG. 12B is a graph of the percent of bound SWI/SNF in 293Tnuclei exposed to stimulated T-cell extract or unstimulated T-cellextract, based on densitometric analysis of duplicate blots. FIG. 12C isa graph of the ATPase activity of the SWI/SNF complex. Followingexposure of 293T nuclei to stimulated T-cell extract or unstimulatedT-cell extract, SWI/SNF was immunoprecipitated from nuclear lysatesusing anti-BRG1 antibodies and hydrolysis of 1 nM exogenous ATP (“ATP+”)by the immune precipitate (“BRG1-IP”) was determined in a luminometricassay. Control precipitations were carried out using pre-immune IgGs(“Pre-I IgG”). ATP levels are expressed as mean (±SD) relative lightunits (RLU). Elevated ATP levels reflect low ATPase activity of theBRG1-IP. FIG. 12D is an immunoblot showing the hyperacetylation of theIL-2 locus in 293T nuclei. MNase-soluble chromatin was prepared frominput 293T nuclei (“Input N”), and the nuclei were exposed tounstimulated T-cell extract or stimulated T-cell extract. Acetylated H4was immunoprecipitated, and DNA was isolated from anti-acH4 precipitate(“Bound”) and supernatant (“Unbound”) fractions. DNA was dot-blotted andhybridized to an IL-2 probe (top rows) and a control β-actin probe(bottom rows). FIG. 12 is a picture of a set of gels showingtranscription of the IL-2 gene. Nuclei from 293T, NT2, and restingT-cells were incubated for two hours in unstimulated T-cell extract(“Nuclei/UTE”) or stimulated T-cell extract (“Nuclei/STE”). As controls,nuclei were incubated in stimulated T-cell extract containing either 100μg/ml RNAse A, mAb414, or 50 nM actinomycin D (“ActD”). RNA was isolatedfrom the reaction mix, and IL-2 transcription was examined by RT-PCR.

FIG. 13 is a graph demonstrating that 293T fibroblasts reprogrammed inthe Jurkat-TAg extract display altered gene expression. Relative mRNAlevels in 293T-cells incubated in stimulated Jurkat-TAg extract or incontrol 293T extract were compared using a cytokine gene expressionarray. Bars represent fold increase or decrease in transcription ofindicated genes in Jurkat extract-treated cells, measured as the ratioof reprogrammed/control probe hybridization signal strength. Genes withan over two-fold increase or decrease in expression level are shown.Different color backgrounds separate distinct gene groups.

FIGS. 14A-14C demonstrate that 293T fibroblasts reprogrammed inJurkat-TAg extract exhibit hematopoietic cell markers and function. FIG.14A is a picture of 293T-cells exposed to a control 293T extract (toprow) or a stimulated Jurkat-TAg extract (middle row), and Jurkat-TAgcells (bottom row) and analyzed by immunofluorescence using indicatedFITC-conjugated antibodies. CD3, CD4, CD8 were detected at four dayspost-reprogramming; CD45 was detected at 11 days post-reprogramming. TheCγ subunit of PKA (“PKA-Cγ”) was examined as a positive control. FIG.14B is a picture of the immunofluorescence analysis of the cells usinganti-TCRαβ antibodies at 11 days post-reprogramming. DNA was labeledwith propidium iodide. FIG. 14C is a picture of each cell typestimulated with anti-CD3 antibodies and PMA for 24 hours, starting attwo days post-reprogramming. Stimulated and unstimulated cells wereanalyzed by immunofluorescence using anti-IL-2Rβ (green) and anti-IL-2Rα(red) antibodies. Stimulation of Jurkat cells and Jurkat extract-treated293T fibroblasts elicited IL-2-Rα synthesis. DNA (blue) was labeled withHoechst 33342. Merged images are shown (bars, 10 μm).

FIG. 15 is a picture of the immunofluorescence analysis of 293Tfibroblasts reprogrammed in NT2 extract, demonstrating that thereprogrammed cells express the neurofilament protein NF-200. NT2 orcontrol 293T extract-treated fibroblasts were examined byimmunofluorescence using anti-NF200 antibodies at 15 dayspost-reprogramming. DNA was labeled with Hoechst 33342 (bar, 10 μm).

FIG. 16A is a set of pictures showing the morphology of NIH3T3fibroblasts and mouse ES cells. FIG. 16B is a set of pictures showingthe morphology of NIH3T3 fibroblasts reprogrammed in mouse embryonicstem cell extract, fibroblasts mock-reprogrammed in NIH3T3 extract, andintact NIH3T3 cells exposed to ES cell extract. Phase contrastmicrographs are shown (bars, 20 μm).

FIGS. 17A and 17B are pictures of the immunofluorescence andimmunoblotting analysis, respectively, of Oct4 in NIH3T3 cells,embryonic stem cells and NIH3T3 fibroblasts reprogrammed in embryonicstem cell extract (“NIH/ES ext.”; day-4 post-reprogramming). NIH3T3cells exposed to a control NIH3T3 cell extract do not express Oct4(“NIH/NIH ext.”).

FIG. 18 is a picture of a membrane showing the detection of alkalinephosphatase activity in mouse embryonic stem cells. The top row containslysates of NIH3T3 cells, and the bottom row contains lysates ofembryonic stem cells. Two and 6 μl lysate were applied onto the membrane(protein concentration is ˜20 μg/μl).

FIG. 19 is a picture of a membrane showing the detection of alkalinephosphatase activity in NIH3T3 cells reprogrammed in the embryonic stemcell extract.

DETAILED DESCRIPTION

We have developed novel methods of reprogramming cells by exposing themor their genetic material to a reprogramming media (e.g., a cellextract). This reprogramming refers to decreasing or eliminating theexpression of genes specific for the donor cell or increasing theexpression of genes specific for another cell type. For example, we haveshown that incubation of nuclei from resting T-cells, B-cells, orfibroblasts in an extract from stimulated T-cells results in migrationof a T-cell specific transcription factor from the extract into thenuclei. Additionally, the reprogramming of nuclei from resting T-cells,fibroblasts, endothelial (HUVEC), differentiated epithelial (HeLa), andneuronal precursor (NT2) cells induced expression of the IL-2 gene, agene that is otherwise repressed by the nuclei. Reprogramming of restingT-cell and fibroblast nuclei also induced hyperacetylation of the IL-2gene and intranuclear anchoring of a chromatin remodeling complex. Thus,reprogramming media such as extracts may be used to alter the expressionprofile of the genetic material of a donor cell such that it resemblesthat of the cells used to prepare the reprogramming media.

The methods for reprogramming a cell that are described herein may beused to convert a cell into another cell-type that is closely related byorigin or character. For example, members of the connective-tissuefamily, such as fibroblasts, smooth muscle cells, osteoblasts,adiopocytes, and chrondrocytes, may be interconverted using thesemethods. Additionally, hepatocytes may be converted intoinsulin-producing B-cells because both of these cell types express manyof the same genes. Alternatively, a cell may be converted into a desiredcell type that is distantly related to the donor cell and thus sharesfew or no characteristics or functions with the donor cell.

In one such reprogramming method, a nucleus from an interphase donorcell is incubated in a reprogramming media prepared from interphasecells (e.g., an interphase cell extract) under conditions that allowexport of factors, such as transcription regulatory factors, from thenucleus and the import of factors from the reprogramming media into thenucleus. The nucleus is then inserted into a recipient cell orcytoplast, forming a reprogrammed cell. Preferably, the cells used toprepare the interphase reprogramming media are the cell type one wishesthe reprogrammed cell to become. Due to the different factors in thenucleus of the reprogrammed cell compared to that of the donor cell, thereprogrammed cell expresses a different set of mRNA and proteinmolecules and thus has a different phenotype than that of the donorcell. To achieve optimum reprogramming efficiency, 2, 3, 5, or morerounds of reprogramming may be performed. The reprogrammed cells mayalso be cultured under conditions promoting sustained changes in cellfunction. For example, the cells may be cultured with additionalcomponents such as antigens, interleukins, growth factors, cytokines, orother cells. The reprogrammed cells can also be transplanted into a hostanimal or patient, in the organ where they are supposed to function.Local environment cues may facilitate reprogramming.

In a related method, the nucleus from a donor interphase cell isincubated in a mitotic reprogramming media (e.g., a mitotic cellextract), a detergent and salt solution, or a protein kinase solution topromote nuclear envelope dissolution and possibly chromatincondensation, forming a chromatin mass. This nuclear envelope breakdownand chromatin condensation facilitate the release of factors from thechromatin mass. Alternatively, a chromatin mass may be isolated from adonor mitotic cell. In one embodiment of this method, the chromatin massis inserted into a recipient cell or cytoplast of the desired cell type.After this nuclear transfer, a nucleus is reformed from the donorchromatin mass. Additionally, desired factors from the cytoplasm of therecipient cell or cytoplast migrate into the nucleus and bind theexogenous chromosomes, resulting in the expression of desired genes bythe reprogrammed cell. To promote the sustained expression of thedesired genes, 2, 3, 5, or more rounds of reprogramming may beperformed, and the reprogrammed cells may also be cultured withadditional components such as antigens, interleukins, growth factors,cytokines, or other cells.

In another embodiment of this method, the chromatin mass is firstincubated in an interphase reprogramming media (e.g., an interphase cellextract) as described above to further promote the release ofundesirable factors from the chromatin mass and the binding of desirablefactors from the interphase reprogramming media to the chromatin mass.The incubation in the interphase reprogramming media also results in theformation of a nuclear membrane, encapsulating the chromatin mass anddesired factors from the reprogramming media. The reformed nucleus isthen inserted into a recipient cell or cytoplast of the desired celltype or of any other cell type.

As an alternative to isolating nuclei or chromatin masses from donorcells for subsequent incubation in a reprogramming media, donor cellsmay be gently permeabilized and incubated in the reprogramming media(e.g., a cell extract). Permeabilization of the plasma membrane allowsfactors to enter and leave the cell. The cells may either be incubatedin an interphase reprogramming media to allow the nucleus to remainmembrane-bounded or with a mitotic reprogramming media to allow thedissolution of the nuclear membrane and formation of a chromatin mass.After incubation in the reprogramming media, the plasma membrane may beresealed, trapping desired factors from the reprogramming media insidethe cell. If desired, this reprogramming method can be repeated 1, 2, 3,4, 5, or more times. For example, after the resealed cell is culturedfor a certain length of time (e.g., after 2 days, 7 days, 14 days, 3weeks, 4 weeks, 8 weeks, or longer) in the presence or absence offactors such as antigens, interleukins, growth factors, cytokines, orother cells to promote reprogramming, the cells are permeabilized andsubjected to an additional round of reprogramming. Additional cycles ofreprogramming may result in more stable and heritable epigenetic changesand in prolonged expression of the phenotype or proteins of interestfrom the reprogrammed cells.

This whole cell reprogramming method was utilized to reprogrampermeabilized, human fibroblast cells using an extract from thelymphoblastic leukemia cell line, Jurkat-TAg (hereafter referred to asJurkat), resulting in the remodeling of chromatin, activation oflymphoid-specific genes, and establishment of a T-cell-specificactivity. For example, T-cell-specific antigens such as the CD3-T-cellreceptor (TCR) complex were expressed by the reprogrammed cells, and theIL-2 receptor was assembled in response to CD3-TCR stimulation of thesecells. After exposure to an NT2 neuronal precursor cell extract,permeabilized fibroblasts expressed a neurofilament protein and extendedneurite-like outgrowths in culture. Fibroblasts were also reprogrammedinto cells resembling embryonic stem cells.

Reprogrammed cells generated from these methods may be used to replacecells in a mammal in need of a particular cell type. The reprogrammingmethods may be used to either directly produce cells of the desired celltype or to produce undifferentiated cells which may be subsequentlydifferentiated into the desired cell type. For example, stem cells maybe differentiated in vitro by culturing them under the appropriateconditions or differentiated in vivo after administration to anappropriate region in a mammal. To optimize phenotypic and functionalchanges, reprogrammed cells can be transplanted into the organ (e.g., aheart) where they are intended to function in an animal model or inhuman patients shortly after reprogramming (e.g., after 1, 2, 3, 5, 7,or more days). Reprogrammed cells implanted in an organ may bereprogrammed to a greater extent than cells grown in culture prior totransplantation. Cells implanted in an animal organ may be removed fromthe organ and transplanted into a recipient mammal such as a human, orthe animal organ may be transplanted into the recipient.

To increase the length of time the cell, nuclei, or chromatin mass maybe reprogrammed in vitro prior to administration to a mammal for thetreatment of disease, the donor cell may be optionally modified by thetransient transfection of a plasmid containing an oncogene flanked byloxP sites for the Cre recombinase and containing a nucleic acidencoding the Cre recombinase under the control of an inducible promoter(Cheng et al., Nucleic Acids Res. 28(24):E108, 2000). The insertion ofthis plasmid results in the controlled immortalization of the cell.After the cell is reprogrammed into the desired cell-type and is readyto be administered to a mammal, the loxP-oncogene-loxP cassette may beremoved from the plasmid by the induction of the Cre recombinase whichcauses site-specific recombination and loss of the cassette from theplasmid. Due to the removal of the cassette containing the oncogene, thecell is no longer immortalized and may be administered to the mammalwithout causing the formation of a cancerous tumor.

Examples of medical applications for these reprogrammed cells includethe administration of neuronal cells to an appropriate area in the humannervous system to treat, prevent, or stabilize a neurological diseasesuch as Alzheimer's disease, Parkinson's disease, Huntington's disease,or ALS; or a spinal cord injury. In particular, degenerating or injuredneuronal cells may be replaced by the corresponding cells from a mammal.This transplantation method may also be used to treat, prevent, orstabilize autoimmune diseases including, but not limited to, insulindependent diabetes mellitus, rheumatoid arthritis, pemphigus vulgaris,multiple sclerosis, and myasthenia gravis. In these procedures, thecells that are attacked by the recipient's own immune system may bereplaced by transplanted cells. In particular, insulin-producing cellsmay be administered to the mammal for the treatment or prevention ofdiabetes, or oligodendroglial precursor cells may be transplanted forthe treatment or prevention of multiple sclerosis. For the treatment orprevention of endocrine conditions, reprogrammed cells that produce ahormone, such as a growth factor, thyroid hormone, thyroid-stimulatinghormone, parathyroid hormone, steroid, serotonin, epinephrine, ornorepinephrine may be administered to a mammal. Additionally,reprogrammed epithelial cells may be administered to repair damage tothe lining of a body cavity or organ, such as a lung, gut, exocrinegland, or urogenital tract. It is also contemplated that reprogrammedcells may be administered to a mammal to treat damage or deficiency ofcells in an organ, muscle, or other body structure or to form an organ,muscle, or other body structure. Desirable organs include the bladder,brain, nervous tissue, esophagus, fallopian tube, heart, pancreas,intestines, gallbladder, kidney, liver, lung, ovaries, prostate, spinalcord, spleen, stomach, testes, thymus, thyroid, trachea, ureter,urethra, and uterus.

Reprogrammed cells may also be combined with a matrix to form a tissueor organ in vitro or in vivo that may be used to repair or replace atissue or organ in a recipient mammal. For example, reprogrammed cellsmay be cultured in vitro in the presence of a matrix to produce a tissueor organ of the urogenital system, such as the bladder, clitoris, corpuscavermosum, kidney, testis, ureter, uretal valve, or urethra, which maythen be transplanted into a mammal (Atala, Curr. Opin. Urol.9(6):517-526, 1999). In another transplant application, synthetic bloodvessels are formed in vitro by culturing reprogrammed cells in thepresence of an appropriate matrix, and then the vessels are transplantedinto a mammal for the treatment or prevention of a cardiovascular orcirculatory condition. For the generation of donor cartilage or bonetissue, reprogrammed cells such as chondrocytes or osteocytes arecultured in vitro in the presence of a matrix under conditions thatallow the formation of cartilage or bone, and then the matrix containingthe donor tissue is administered to a mammal. Alternatively, a mixtureof the cells and a matrix may be administered to a mammal for theformation of the desired tissue in vivo. Preferably, the cells areattached to the surface of the matrix or encapsulated by the matrix.Examples of matrices that may be used for the formation of donor tissuesor organs include collagen matrices, carbon fibers, polyvinyl alcoholsponges, acrylateamide sponges, fibrin-thrombin gels, hyaluronicacid-based polymers, and synthetic polymer matrices containingpolyanhydride, polyorthoester, polyglycolic acid, or a combinationthereof (see, for example, U.S. Pat. Nos. 4,846,835; 4,642,120;5,786,217; and 5,041,138).

These methods are described further below. It is noted that any of themethods described below can be performed with reprogramming media otherthan cell extracts. For example, a reprogramming media can be formed byadding one or more naturally-occurring or recombinant factors (e.g.,nucleic acids or proteins such as DNA methyltransferases, histonedeacetylases, histones, nuclear lamins, transcription factors,activators, repressors, growth factors, hormones, or cytokines) to asolution, such as a buffer. Preferably, one or more of the factors arespecific for the cell type one wishes the donor cell to become.

Example 1 One-Step In Vitro Reprogramming Method

In the following method for reprogramming cells, nuclei are isolatedfrom interphase cells and incubated in an interphase reprogramming media(e.g., an interphase cell extract) under conditions that allow theaddition of factors from the reprogramming media to the nuclei or theremoval of factors from the nuclei. Preferably, the nuclei remainmembrane-bounded during this incubation. The reprogrammed nuclei arethen isolated from the reprogramming media and inserted into recipientcells or cytoplasts.

Isolation of Nuclei

Preferably, cells from the subject who will receive the reprogrammedcells are used as the source of donor nuclei. However, cells from othermembers of the same species or members of a different species or genusmay be used. As many as several million nuclei may be isolated fromsynchronized or unsynchronized cell populations in culture. The cellpopulations may be synchronized naturally or chemically. Preferably, atleast 40, 60, 80, 90, or 100% of the cells in a population are arrestedin interphase, such as in one or more of the following phases of thecell cycle: G_(o), G₁, S, or G₂, using standard procedures.

To accomplish this, cells may be incubated, for example, in low serum,such as 5%, 2%, or 0% serum, for 1, 2, 3, or more days to increase thepercentage of cells in G_(o) phase. To synchronize cells in G₁, thecells may be grown to confluence as attached cells and then incubated in0.5-1 μg/ml nocodazole (Sigma Chemicals, St. Louis, Mo.) for 17-20hours, as described previously (see, for example, Collas et al., 1999and references therein). The flasks containing the attached cells areshaken vigorously by repeatedly tapping the flasks with one hand,resulting in the detachment of mitotic cells and G₁ phase doublets. TheG₁ phase doublets are pairs of elongated cells at the end of thedivision process that are still connected by a thin bridge. Detached G₁phase doublets may be isolated from the media based on thischaracteristic doublet structure. The G₁ phase doublets may remainattached or may divide into two separate cells after isolation.

To increase the percentage of cells in S phase, the cells may becultured in the presence of aphidicolin which inhibits DNA polymerase-αand thus inhibits DNA synthesis and arrests cells in S phase.Alternatively, cells may be incubated in the presence of excessthymidine. The resulting high intracellular concentration of thymidinerelative to that of other nucleotides also inhibits DNA polymerase.

Cells may be synchronized in G₂ by incubating the cells in the presenceof aphidicolin to arrest them in S phase and then washing the cellsthree times by repeated centrifugation and resuspension in phosphatebuffered saline (PBS), as described herein. The cells are then incubatedfor a length of time sufficient for cells to enter G₂ phase. Forexample, cells with a doubling time of approximately 24 hours, may beincubated for between 6 and 12 hours to allow them to enter G₂ phase.For cells with shorter or longer doubling times, the incubation time maybe adjusted accordingly.

The synchronized or unsynchronized cells are harvested in PBS usingstandard procedures, and several washing steps are performed to transferthe cells from their original media into a hypotonic buffer (10 mMHepes, pH 7.5, 2 mM MgCl₂, 25 mM KCl, 1 mM DTT, 10 μM aprotinin, 10 μMleupeptin, 10 μM pepstatin A, 10 μM soybean trypsin inhibitor, and 100μM PMSF). For example, the cells may be washed with 50 ml of PBS andpelleted by centrifugation at 500×g for 10 minutes at 4° C. The PBSsupernatant is decanted, and the pelleted cells are resuspended in 50 mlof PBS and centrifuged, as described above. After this centrifugation,the pelleted cells are resuspended in 20-50 volumes of ice-coldhypotonic buffer and centrifuged at 500×g for 10 minutes at 4° C. Thesupernatant is again discarded and approximately 20 volumes of hypotonicbuffer are added to the cell pellet. The cells are carefully resuspendedin this buffer and incubated on ice for at least one hour, resulting inthe gradual swelling of the cells.

To allow isolation of the nuclei from the cells, the cells are lysedusing standard procedures. For example, 2-5 ml of the cell suspensionmay be transferred to a glass homogenizer and Dounce homogenized usingan initial 10-20 strokes of a tight-fitting pestle. Alternatively, thecell suspension is homogenized using a motorized mixer (e.g.,Ultraturrax). If desired, cell lysis may be monitored using phasecontrast microscopy at 40-fold magnification. During thishomogenization, the nuclei should remain intact and most or preferablyall of the originally attached cytoplasmic components such as vesicles,organelles, and proteins should be released from the nuclei. Ifnecessary, 1-20 μg/ml of the cytoskeletal inhibitors, cytochalasin B orcytochalasin D, may be added to the aforementioned hypotonic buffer tofacilitate this process. Homogenization is continued as long asnecessary to lyse the cells and release cytoplasmic components from thenuclei. For some cell types, as many as 100, 150, or more strokes may berequired. The lysate is then transferred into a 15 ml conical tube onice, and the cell lysis procedure is repeated with the remainder of thesuspension of swollen cells. Sucrose from a 2 M stock solution made inhypotonic buffer is added to the cell lysate, resulting in a finalconcentration of 250 mM sucrose. This solution is mixed by inversion,and the nuclei are pelleted by centrifugation at 400×g in a swing outrotor for 10 to 40 minutes at 4° C. The supernatant is then discarded,and the pelleted nuclei are resuspended in 10-20 volumes of nuclearbuffer (10 mM Hepes, pH 7.5, 2 mM MgCl₂, 250 mM sucrose, 25 mM KCl, 1 mMDTT, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10 μM soybeantrypsin inhibitor, and 100 μM PMSF). The nuclei are sedimented andresuspended in 1-2 volumes of nuclear buffer, as described above. Thefreshly isolated nuclei may either be used immediately for in vitroreprogramming and nuclear transfer into recipient cells or cytoplasts asdescribed below or stored for later use. For storage, the nuclei arediluted in nuclear buffer to a concentration of approximately 10⁶/ml.Glycerol (2.4 volumes of 100% glycerol) is added and mixed well bygentle pipetting. The suspension is aliquoted into 100-500 μl volumes in1.5-ml tubes on ice, immediately frozen in a methanol-dry ice bath, andstored at −80° C. Prior to use, aliquots of the nuclei are thawed on iceor at room temperature. One volume of ice cold nuclear buffer is added,and the solution is centrifuged at 1,000×g for 15 minutes in a swing outrotor. The pelleted nuclei are resuspended in 100-500 μl nuclear bufferand centrifuged as described above. The pelleted nuclei are thenresuspended in a minimal volume of nuclear buffer and stored on iceuntil use.

Preparation of the Reprogramming Extract

Interphase cultured cells as harvested using standard methods and washedby centrifugation at 500×g for 10 minutes in a 10 ml conical tube at 4°C. Preferably, the cells are of the desired cell type that one wishesthe recipient cell or cytoplast to become. The supernatant is discarded,and the cell pellet is resuspended in a total volume of 50 ml of coldPBS. The cells are centrifuged at 500×g for 10 minutes at 4° C. Thiswashing step is repeated, and the cell pellet is resuspended inapproximately 20 volumes of ice-cold interphase cell lysis buffer (20 mMHepes, pH 8.2, 5 mM MgCl₂, 1 mM DTT, 10 μM aprotinin, 10 μM leupeptin,10 μM pepstatin A, 10 μM soybean trypsin inhibitor, 100 μM PMSF, andoptionally 20 μg/ml cytochalasin B). The cells are sedimented bycentrifugation at 800×g for 10 minutes at 4° C. The supernatant isdiscarded, and the cell pellet is carefully resuspended in no more thanone volume of interphase cell lysis buffer. The cells are incubated onice for one hour to allow swelling of the cells. The cells are lysed byeither sonication using a tip sonicator or Dounce homogenization using aglass mortar and pestle. Cell lysis is performed until at least 90% ofthe cells and nuclei are lysed, which may be assessed using phasecontrast microscopy. The sonication time required to lyse at least 90%of the cells and nuclei may vary depending on the type of cell used toprepare the extract.

The cell lysate is placed in a 1.5-ml centrifuge tube and centrifuged at10,000 to 15,000×g for 15 minutes at 4° C. using a table top centrifuge.The tubes are removed from the centrifuge and immediately placed on ice.The supernatant is carefully collected using a 200 μl pipette tip, andthe supernatant from several tubes is pooled and placed on ice. Thissupernatant is the “interphase cytoplasmic” or “IS15” extract. This cellextract may be aliquoted into 20 μl volumes of extract per tube on iceand immediately flash-frozen on liquid nitrogen and stored at −80° C.until use. Alternatively, the cell extract is placed in anultracentrifuge tube on ice (e.g., fitted for an SW55 Ti rotor;Beckman). If necessary, the tube is overlayed with mineral oil to thetop. The extract is centrifuged at 200,000×g for three hours at 4° C. tosediment membrane vesicles contained in the IS15 extract. At the end ofcentrifugation, the oil is discarded. The supernatant is carefullycollected, pooled if necessary, and placed in a cold 1.5 ml tube on ice.This supernatant is referred to as “IS200” or “interphase cytosolic”extract. The extract is aliquoted and frozen as described for the IS15extract.

If desired, the extract can be enriched with additional nuclear factors.For example, nuclei can be purified from cells of the cell type fromwhich the reprogramming extract is derived and lysed by sonication asdescribed above. The nuclear factors are extracted by a 10-60 minuteincubation in nuclear buffer containing NaCl or KCl at a concentrationof 0.15-800 mM under agitation. The lysate is centrifuged to sedimentunextractable components. The supernatant containing the extractedfactors of interest is dialyzed to eliminate the NaCl or KCl. Thedialyzed nuclear extract is aliquoted and stored frozen. This nuclearextract is added at various concentrations to the whole cell extractdescribed above prior to adding the nuclei for reprogramming.

As an alternative to a cell extract, a reprogramming media can also beformed by adding one or more naturally-occurring or recombinant factors(e.g., nucleic acids or proteins such as DNA methyltransferases, histonedeacetylases, histones, nuclear lamins, transcription factors,activators, repressors, growth factors, hormones, or cytokines) to asolution, such as a buffer. Preferably, one or more of the factors arespecific for the cell type one wishes the donor cell to become.

Reprogramming of Nuclei in the Extract

Either freshly isolated or thawed purified nuclei are resuspended in thereprogramming media described in the previous section at a concentrationof 4,000-5,000 nuclei/μl. An ATP generating system (1 mM ATP, 10 mMcreatine phosphate, 25 μg/ml creatine kinase) and 100 μM GTP are addedto the interphase extract to promote active uptake of nuclear componentsby the exogenous nuclei. The reaction is incubated at 30° C. for up totwo hours. Uptake of specific nuclear components may be monitored byimmunofluorescence analysis of the nuclei, as shown in FIGS. 3A and 6A.

Purification of Reprogrammed Nuclei Out of the Extract

The reprogrammed nuclei are centrifuged at 1,000×g for 10-30 minutesthrough a 1 M sucrose cushion prepared in nuclear buffer at 4° C. Thenuclei are washed by resuspending them in 500 μl cold nuclear buffer andcentrifuging at 1,000×g for 10 minutes at 4° C. The nuclei areresuspended in nuclear buffer and held on ice until use for nucleartransfer into the cytoplasm of recipient cells or cytoplasts.

Enucleation of Recipient Cells

Preferably, part or all of the DNA in the recipient cell is removed orinactivated. This destruction or removal of the DNA in the recipientcell prevents the genetic material of the cell from contributing to thecharacteristics and function of the reprogrammed cell. One method fordestroying the nucleus of the cell is exposure to ultraviolet light(Gurdon, in Methods in Cell Biology, Xenopus Laevis: —Practical Uses incell and Molecular Biology, Kay and Peng, eds., Academic Press,California, volume 36: pages 299-309, 1991). Alternatively, the nucleusmay be surgically removed by any standard technique (see, for example,McGrath and Solter, Science 220:1300-1319, 1983). In one possiblemethod, a needle is placed into the cell, and the nucleus is aspiratedinto the inner space of the needle. The needle may then be removed fromthe cell without rupturing the plasma membrane (U.S. Pat. Nos. 4,994,384and 5,057,420).

Introduction of Reprogrammed Nuclei into Recipient Cells or Cytoplasts

The nuclei are introduced into recipient cells or cytoplasts of eitherthe desired cell type or of any other cell type using standard methods,such as microinjection or electrofusion (see, for example, U.S. Pat.Nos. 4,997,384 and 5,945,577). The reconstituted cells are placed backin culture and allowed to recover, divide, and differentiate accordingto the reprogrammed pathway. Gene expression by the reprogrammed cellsmay be monitored using standard Northern analysis to measure expressionof mRNA molecules, preferably mRNA molecules that are specific for thedonor cell, recipient cell, or the desired cell type (Ausubel et al.,supra). Expression of specific mRNA molecules may also be detected usingstandard reverse-transcription polymerase chain reaction (RT-PCR) assayswith primers designed to specifically bind an mRNA molecule of interest.Alternatively, the expression of multiple cell specific mRNA moleculesmay be monitored using standard DNA chip technology with cDNA arrays(Marrack et al., Current Opinion in Immunology 12, 206-209, 2000;Harkin, Oncologist. 5:501-507, 2000; Pelizzari et al., Nucleic AcidsRes. 2; 28(22):4577-4581, 2000; Marx, Science 289(5485):1670-1672,2000). The cells may be analyzed for a reduction in expression of genesspecific for the cell type of the donor cell, recipient cell, orrecipient cytoplast. Additionally, cells may be assayed for an increasein expression of genes specific for the desired cell type. Examples ofmRNA molecules that are indicative of reprogramming to generate a stemcell include H-19, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, Oct-4,Genesis, GCNF, GDF-3, and TDGF-1. Neural cell specific mRNA moleculesinclude, but are not limited to, NGF, NF-H, NeuN, NSE, and CD11b. Foranalyzing the conversion to an adipocyte cell fate, expression of mRNAmolecules such as leptin, PPARλ1, PPARλ2, SREBP1C, IR, and TNFα may bemonitored. IGF-1 and IR are indicative of insulin producing cells.Additionally, the cells may be analyzed for expression of particularproteins using standard Western or immunofluorescence analysis (Ausubelet al., supra).

Examples of other characteristics of the reprogrammed cell that may beanalyzed to determine whether it has been converted into the desiredcell type include the size of the cell, cell morphology, ability to growas an adherent cell, ability to grow as an attached cell, volume ofcytoplasm, and location of a centrosome. The functions of thereprogrammed cells may also be tested, such as the ability of red bloodcells to transport O₂ and CO₂, the ability of B-cells to makeantibodies, and the ability of neutrophiles to phagocytose and destroyinvading bacteria. Additionally, the production of lipids by adicotypesmay be determined using standard microscopy to visualize lipid dropletsin the cells.

Example 2 Two-Step In Vitro Reprogramming Method

In another method for reprogramming cells, nuclei are isolated frominterphase cells and incubated in a mitotic extract, a detergent andsalt solution, or a protein kinase solution to induce nuclear envelopebreakdown and the formation of chromatin masses. This incubation causesthe release of factors from the chromatin masses. Alternatively,chromatin masses may be isolated from mitotic cells. Preferably, thechromatin masses are then incubated in an interphase reprogrammingextract to promote the formation of nuclear membranes and the additionof desired factors from the extract to the resulting nuclei. Thereprogrammed nuclei are then isolated from the extract and inserted intorecipient cells or cytoplasts of the desired cell type or of any othercell type.

Alternatively, the chromatin masses may be directly inserted intorecipient cells or cytoplasts without first being induced to reformnuclei. For this embodiment, recipient cells or cytoplasts of thedesired cell type are used so that desired factors from the cytoplasm ofthe recipient cells or cytoplasts may bind the exogenous chromosomesfrom the donor chromatin masses and further promote the expression ofdesired mRNA and protein molecules.

Preparation of Mitotic Cell Extract

A mitotic cytoplasmic (MS15) or mitotic cytosolic (MS200) extract may beprepared as described above for interphase IS15 or IS200 extract, exceptthat mitotic cells are used instead of interphase cells and that 10 mMEDTA is added to the cell lysis buffer. If desired, the extract can beenriched with additional nuclear factors as described in Example 1. Forthe isolation of mitotic cells, somatic cells are synchronized inmitosis by incubating them in 0.5-1 μg/ml nocodazole for 17-20 hours,and the mitotic cells are detached by vigorous shaking, as describedabove. The detached G₁ phase doublets may be discarded, or they may beallowed to remain with the mitotic cells which constitute the majority(over 80%) of the detached cells. The harvested detached cells arecentrifuged at 500×g for 10 minutes in a 10 ml conical tube at 4° C.

Chromosome Condensation Reaction in Mitotic Extract for Removal ofEndogenous Nuclear Components

An aliquot of MS15 or MS200 extract is thawed on ice. An ATP-generatingsystem (0.6 μl) and GTP are added to 20 μl of extract and mixed byvortexing, resulting in final concentrations of 1 mM ATP, 10 mM creatinephosphate, 25 μg/ml creatine kinase, and 100 μM GTP.

Nuclei are isolated from donor cells as described above. The nucleisuspension is added to the extract at a concentration of 1 μl nuclei per10 μl of extract, mixed well by pipetting, and incubated in a 30, 33,35, 37, or 39° C. water bath. The tube containing the mixture is tappedgently at regular intervals to prevent chromosomes from clumping at thebottom of the tube. Nuclear envelope breakdown and chromosomecondensation is monitored at regular intervals, such as every 15minutes, under a microscope. When the nuclear envelope has broken downand chromosomes have started to condense, the procedure for recovery ofchromatin masses from the extract is started.

Formation of Decondensed Chromatin Masses by Exposure of Nuclei toMitotic Extract and Anti-NuMA Antibodies

Alternatively, chromatin masses that are not condensed or only partiallycondensed may be formed by performing the above procedure afterpre-loading the isolated nuclei with an antibody to the nuclear matrixprotein NuMA (Steen et al., J. Cell Biol. 149, 531-536, 2000). Thisprocedure allows the removal of nuclear components from chromatin by thedissolution of the nuclear membrane surrounding the donor nuclei;however, the condensation step is inhibited by addition of the anti-NuMAantibody. Preventing chromosome condensation may reduce the risk ofchromosome breakage or loss while the chromosomes are incubated in themitotic extract.

For this procedure, purified cell nuclei (2,000 nuclei/μl) arepermeabilized in 500 μl nuclear buffer containing 0.75 μg/mllysolecithin for 15 minutes at room temperature. Excess lysolecithin isquenched by adding 1 ml of 3% BSA made in nuclear buffer and incubatingfor 5 minutes on ice. The nuclei are then sedimented and washed once innuclear buffer. The nuclei are resuspended at 2,000 nuclei/μl in 100 μlnuclear buffer containing an anti-NuMA antibody (1:40 dilution;Transduction Laboratories). After a one hour incubation on ice withgentle agitation, the nuclei are sedimented at 500×g through 1 M sucrosefor 20 minutes. The nuclei are then resuspended in nuclear buffer andadded to a mitotic extract containing an ATP regenerating system, asdescribed in the previous section. Optionally, the anti-NuMA antibodymay be added to the extract to further prevent chromosome condensation.

Formation of Decondensed Chromatin Masses by Exposure of Nuclei to aDetergent or Protein Kinase

Chromatin masses that are not condensed or only partially condensed mayalso be formed by exposure to a detergent or protein kinase. A detergentmay be used to solubilize nuclear components that are either unbound orloosely bound to the chromosomes in the nucleus, resulting in theremoval of the nuclear envelope. For this procedure, purified cellnuclei (2,000-10,000 nuclei/μl) are incubated in nuclear buffersupplemented with a detergent, such as 0.1% to 0.5% Triton X-100 orNP-40. To facilitate removal of the nuclear envelope, additional salt,such as NaCl, may be added to the buffer at a concentration ofapproximately 0.1, 0.15, 0.25, 0.5, 0.75, or 1 M. After a 30-60 minuteincubation on ice with gentle shaking, the nuclei are sedimented bycentrifugation at 1,000×g in a swing-out rotor for 10-30 minutes,depending on the total volume. The pelleted nuclei are resuspended in0.5 to 1 ml nuclear buffer and sedimented as described above. Thiswashing procedure is repeated twice to ensure complete removal of thedetergent and extra salt.

Alternatively, the nuclear envelope may be removed using recombinant ornaturally-occurring protein kinases, alone or in combination.Preferably, the protein kinases are purified using standard proceduresor obtained in purified form from commercial sources. These kinases mayphosphorylate components of the nuclear membrane, nuclear matrix, orchromatin, resulting in removal of the nuclear envelope (see, forexample, Collas and Courvalin, Trends Cell Biol. 10: 5-8, 2000).Preferred kinases include cyclin-dependent kinase 1 (CDK1), proteinkinase C (PKC), protein kinase A (PKA), MAP kinase, andcalcium/calmodulin-dependent kinase (CamKII). For this method,approximately 20,000 purified nuclei are incubated in 20 μl ofphosphorylation buffer at room temperature in a 1.5 ml centrifuge tube.A preferred phosphorylation buffer for CDK1 (Upstate Biotechnology)contains 200 mM NaCl, 50 mM Tris-HCl (pH 7.2-7.6), 10 mM MgSO₄, 80 mMβ-glycerophosphate, 5 mM EGTA, 100 μM ATP, and 1 mM DTT. For PKC, apreferred buffer contains 200 mM NaCl, 50 mM Tris-HCl (pH 7.2-7.6), 10mM MgSO₄, 100 μM CaCl₂, 40 μg/ml phosphatidylserine, 20 μMdiacylglycerol, 100 μM ATP, and 1 mM DTT. If both PKC and CDK1 are usedsimultaneously, the CDK1 phosphorylation buffer supplemented with 40μg/ml phosphatidylserine and 20 μM diacylglycerol is used. A preferredphosphorylation buffer for PKA includes 200 mM NaCl, 10 mM MgSO4, 10 mMTris, pH 7.0, 1 mM EDTA, and 100 μM ATP. For MAP kinase, the PKAphosphorylation buffer supplemented with 10 mM CaCl₂, and 1 mM DTT maybe used. For CamKII, either PKA buffer supplemented with 1 mM DTT or aCam Kinase assay kit from Upstate Biotechnology (Venema et al. J. Biol.Chem 272: 28187-90, 1997) is used.

The phosphorylation reaction is initiated by adding a protein kinase toa final amount of 25-100 ng. The reaction is incubated at roomtemperature for up to one hour. Nuclear envelope breakdown may bemonitored by microscopy during this incubation, such as at 15 minuteintervals. After nuclear envelope breakdown, nuclei are washed threetimes, as described above for the removal of the detergent solution.

Recovery of Chromatin Masses from the Extract, Detergent and SaltSolution, or Protein Kinase Solution

The extract or solution containing the condensed, partially condensed,or not condensed chromatin masses is placed under an equal volume of 1 Msucrose solution made in nuclear buffer. The chromatin masses aresedimented by centrifugation at 1,000×g for 10-30 minutes depending onthe sample volume in a swing out rotor at 4° C. The supernatant isdiscarded and the pelleted chromatin masses are carefully resuspended bypipetting in 0.1-1.0 ml nuclear buffer and centrifuged at 1,000×g for10-30 minutes. The supernatant is discarded, and the pelleted chromatinmasses are resuspended in nuclear buffer and stored on ice until use.

Isolation of Chromatin Masses from Mitotic Cells

As an alternative to generating chromatin masses by exposure of nucleito a mitotic extract, a detergent and salt solution, or a protein kinasesolution, chromatin masses may be obtained by lysis of cellssynchronized in mitosis and centrifugation of the cell lysate asdescribed herein.

Preparation of Membrane Vesicles for Nuclear Reassembly In Vitro

The pellet generated from the 200,000×g centrifugation during thepreparation of the MS2000 mitotic extract is used as a source of mitoticmembrane vesicles. This pellet is resuspended in membrane wash buffer(250 mM sucrose, 50 mM KCl, 2.5 mM MgCl₂, 50 mM Hepes, pH 7.5, 1 mM DTT,1 mM ATP, 10 μM aprotinin, 10 μM leupeptin, 10 μM pepstatin A, 10 μMsoybean trypsin inhibitor, and 100 μM PMSF), centrifuged at 100,000×gfor 30 minutes, aliquoted, frozen in liquid nitrogen, and stored at −80°C.

Nuclear Reassembly Assay

If desired, nuclei may be reassembled from condensed, partiallycondensed, or decondensed chromatin masses as described below. Thereformation of the nuclear membrane around the chromosomes mayencapsulate factors from the extract used for reassembly allowing themto be transferred as part of the reformed nucleus into the recipientcell or cytoplast. The chromatin masses are recovered by sedimentationthrough a 1 M sucrose cushion and are resuspended in interphase extractat a concentration of 4,000-5,000 chromatin masses/μl. Preferably, thisinterphase extract is formed from cells of the cell type that isdesired, as described above. The extract is supplemented with membranevesicles prepared as described above to provide membranes which arerequired for nuclear envelope assembly. The membranes are added at aconcentration of 1 μl thawed membranes per 10 μl extract and mixed byvortexing. An ATP generating system (2 mM ATP, 20 mM creatine phosphate,50 μg/ml creatine kinase) and 100 μM GTP are added to the interphaseextract to promote chromatin decondensation, binding of nuclear membranevesicles to chromatin, and vesicle fusion to form an intact nuclearmembrane. The reaction is incubated at 30° C. for up to two hours, andnuclear reassembly is monitored by phase contract microscopy.

Purification of Reprogrammed Nuclei Out of the Extract

Reprogrammed nuclei are centrifuged at 1,000×g for 10-30 minutes througha 1 M sucrose cushion prepared in nuclear buffer at 4° C. The nuclei arewashed by resuspending them in 500 μl cold nuclear buffer andsedimentation at 1,000×g for 10 minutes at 4° C. Then, the nuclei areresuspended in nuclear buffer and held on ice until use for nucleartransfer into the cytoplasm of recipient cells or cytoplasts.

Introduction of Reprogrammed Nuclei or Chromatin Masses into RecipientCells or Cytoplasts

The chromatin masses or nuclei formed from the chromatin masses areinserted into recipient cells or cytoplasts using standard methods, andgene expression is monitored, as described above.

Example 3 Reprogramming of Permeabilized Cells without Nuclear Transfer

Cells may also be reprogrammed without requiring the isolation of nucleior chromatin masses from the cells. In this method, interphase ormitotic cells are permeabilized and then incubated in an interphase ormitotic reprogramming extract under conditions that allow the exchangeof factors between the extract and the cells. If an interphase extractis used, the nuclei in the cells remain membrane-bounded; if a mitoticextract is used, nuclear envelope breakdown and chromatin condensationmay occur. After the nuclei are reprogrammed by incubation in thisextract, the plasma membrane is preferably resealed, forming an intactreprogrammed cell that contains desired factors from the extract. Ifdesired, the extract can be enriched with additional nuclear factors asdescribed in Example 1.

Permeabilization of Cells

Cells that may be reprogrammed using this procedure includeunsynchronized cells and cells synchronized in G_(o), G₁, S, G₂, or Mphase or a combination of these phases. The cells are permeabilizedusing any standard procedure, such as permeabilization with digitonin orStreptolysin O. Briefly, cells are harvested using standard proceduresand washed with PBS. For digitonin permeabilization, cells areresuspended in culture medium containing digitonin at a concentration ofapproximately 0.001-0.1% and incubated on ice for 10 minutes. Forpermeabilization with Streptolysin O, cells are incubated inStreptolysin O solution (see, for example, Maghazachi et al., 1997 andreferences therein) for 15-30 minutes at room temperature. After eitherincubation, the cells are washed by centrifugation at 400×g for 10minutes. This washing step is repeated twice by resuspension andsedimentation in PBS. Cells are kept in PBS at room temperature untiluse. Alternatively, the cells can be permeabilized while placed oncoverslips as described in Example 6 to minimize the handling of thecells and to eliminate the centrifugation of the cells, therebymaximizing the viability of the cells. Preferably, the cells areimmediately added to the interphase or mitotic extract forreprogramming, as described below.

Reprogramming of Cells in Extract

An interphase or mitotic extract is prepared as described above,preferably using cells of the cell type that one desires thepermeabilized cells to become. The permeabilized cells are suspended inthe reprogramming extract at a concentration of approximately 100-1,000cells/μl. The ATP generating system and GTP are added to the extract asdescribed above, and the reaction is incubated at 30-37° C. for up totwo hours to promote translocation of factors from the extract into thecell and active nuclear uptake or chromosome-binding of factors. Thereprogrammed cells are centrifuged at 800×g, washed by resuspension, andcentrifugation at 400×g in PBS. The cells are resuspended in culturemedium containing 20-30% fetal calf serum (FCS) and incubated for 1-3hours at 37° C. in a regular cell culture incubator to allow resealingof the cell membrane. The cells are then washed in regular warm culturemedium (10% FCS) and cultured further using standard culturingconditions.

Example 4 Reprogramming Using an Activated T-Cell Extract

This reprogramming study using an activated T-cell extract is based onfunctional differences between resting and activated T-cells.Antigen-mediated activation of resting peripheral blood T-cells bystimulation of the T-cell antigen receptor-CD3 (TCR-CD3) complex and theCD28 co-stimulatory receptor induces chromatin remodeling and activationof numerous genes. One such gene is the T-cell-specific growth factorinterleukin-2 (IL-2) gene. IL-2 regulation involves thestimulation-dependent activators NFAT, NFκB, AP-1, the constitutivetranscription factor Oct-1, and the mitogen-activated protein kinase,Erk.

Cell extracts from activated human T-cells were used to induce nuclearlocalization of transcription factors in unactivated human T-cells,B-cells, human fibroblasts, and HeLa cells. Additionally, thisincubation promoted DNA-binding of the chromatin remodeling SWI/SNFcomplex, hyperacetylation of the IL-2 gene and promoter, and expressionof IL-2 mRNA in unactivated T-cells. Expression of IL-2 was also inducedin primary vascular endothelial cells, epithelial cells, and neuronalprecursor cells.

To demonstrate that activation of intact T-cells induces expression ofIL-2, human T-cells were purified from peripheral blood, culturedovernight, and stimulated with anti-CD3 antibodies (clone SpvT3dobtained from A. M. Rasmussen, Norwegian Radium Hospital, Montebello,Norway). In particular, T-cells were purified from peripheral blood fromhealthy donors (Skalhegg et al., Science 263:84-87, 1994). Cells werecultured for 20 hours and incubated on ice for 15 minutes at 5-10×10⁷cells/ml in RPMI1640 (Gibco BRL). The TCR-CD3 complex was stimulatedwith 5 μg/ml anti-CD3 antibodies, and the cells were incubated on icefor 30 minutes. Cells were spun at 400×g at 4° C. for 7 minutes, washed,and resuspended to 5×10⁷ cells/ml in ice-cold RPMI1600. An anti-mouseFab fragment (10 μg/ml) was added as a cross-linker, and the cells wereincubated at 37° C. (t=0 min post-stimulation). At the indicated timepoints, cells were diluted with ice-cold PBS, snap-frozen in liquidnitrogen, thawed, and washed in PBS. Total RNA was isolated, and RT-PCRwas performed using IL-2-specific primers. As illustrated in FIG. 1,IL-2 mRNA is expressed in activated T-cells but not expressed inmock-treated T-cells (“30c,” “60c,” and “120c” denote mock-treatedcells).

To determine whether extract from activated T-cells can increase nuclearlocalization of transcription factors in other cells, T-cells wereactivated by incubation in the presence of an anti-CD3 antibody(Skalhegg, et al., Science 263:84-87, 1994), and then the cells werewashed to remove the unbound antibody. Stimulated T-cell extracts (SE)were prepared 5-10 minutes after anti-CD3 stimulation (i.e., 2 hoursbefore onset of transcription of the IL-2 gene). This timing forpreparation of the extract allowed unequivocal detection of IL-2transcription in the reprogrammed nuclei because the stimulated extractdid not contain any endogenous IL-2 mRNA. To halt all reactions, cellswere snap-frozen in liquid nitrogen at 5-10 minutes post-stimulation,thawed, washed in ice-cold PBS and in lysis buffer (10 mM Hepes, pH 8.2,50 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and protease inhibitors), andsedimented. The pellets were resuspended in two volumes of lysis buffer.

A stimulated T-cell extract was prepared by lysing these T-cells,centrifuging them at 15,000×g, isolating the supernatant, and adding anATP-generating system to the supernatant. In particular, cells weredisrupted with a tip sonicator until over 90% of the cells and nucleiwere lysed. The lysate was cleared at 15,000×g for 15 minutes at 4° C.The supernatant was used fresh or aliquoted, frozen in liquid nitrogen,and stored at −80° C. This simple method does not require dialysis, andtherefore the extract remains concentrated (˜25 mg/ml protein), and theprocedure minimizes risks of proteolysis. Extract from unstimulatedT-cells (USE) were prepared from mock (H₂O)-stimulated T-cells. Areprogramming reaction consisted of 20 μl or multiples thereof ofstimulated extract or unstimulated extract containing 10⁵ nuclei and anATP generating system (1 mM ATP, 10 mM creatine phosphate, 25 μg/mlcreatine kinase, and 100 μM GTP).

To generate donor nuclei, resting peripheral blood T-cells were washedand resuspended in 20 volumes of ice-cold hypotonic nuclear buffer (10mM Hepes, pH 7.5, 2 mM MgCl₂, 25 mM KCl, 1 mM DTT, and proteaseinhibitors). Nuclei were isolated by careful Dounce-homogenization,sedimented at 400×g and washed in nuclear buffer (hypotonic nuclearbuffer/250 mM sucrose). HUVEC, HeLa, and NT2 nuclei were isolatedsimilarly. Nuclear integrity prior to, and after, incubation in theextract was monitored by phase contrast microscopy and by nuclearmembrane labeling with 10 μg/ml of the lipophilic dye, DiOC₆ (FIG. 8A).Nuclei purified from resting T-cells, from the B-cell line Reh, 293Tfibroblasts, or HeLa cells were incubated in this extract for 30 minutesat a concentration of approximately 5,000 nuclei per μl of extract andat 30° C. unless otherwise indicated. Then, the nuclei were purified bysedimentation for 10 minutes through a 1 M sucrose cushion.Alternatively, RNA was extracted from the reaction mix for RT-PCR. Asdemonstrated by immunofluorescence analysis, the T-cell specifictranscription factor NFAT was imported into the nuclei exposed to thestimulated extract (FIGS. 3A and 6A).

The ability of other transcription factors from the extract to migrateinto the nuclei of T-cells, B-cells, fibroblasts, and HeLa cells wasalso determined. For this assay, input donor nuclei (“Input”) fromunstimulated T-cells were incubated in either stimulated extract (“SE”),control extract prepared from unstimulated T-cells (denoted “USE” forunstimulated extract), or stimulated extract containing a monoclonalantibody against nucleoporins which sterically blocks nuclear import(“SE+mAb414”). The nuclei were then purified from the extract bycentrifugation and resuspension. As expected for a whole cell extract,NFAT, AP-1, or NFκB, Oct-1 and Erk (1 and 2) were detected on Westernblots of input stimulated extract prior to incubation of the nuclei(FIG. 8B). Virtually no AP-1 was seen in input unstimulated extractlikely because the complex is not assembled in unstimulated T-cells, andno NFAT, AP-1, NFκB and little Erk were detected in input nuclei (FIG.8B). As illustrated in FIGS. 3B and 8B, T-cell nuclei incubated in thestimulated extract had increased levels of NFAT, c-Jun/AP1, NFκB, andMAP kinase (Erk1 and Erk2), as measured using standard Western blotanalysis with an anti-histone H4 antibody as a loading control and withantibodies to each factor (e.g., the anti-Erk antibody obtained from DrJ. Kubiak, CNRS, Paris, France). The AP-1 transcription complex was alsoassembled in the nuclei (FIG. 8B), presumably as a result of Jun-Fosassociation. And Erk was imported into nuclei exposed to stimulatedextract (FIG. 8B). Nuclear import of all factors was verified byimmunofluorescence analysis. Nuclear uptake of these factors in restingT-cell nuclei occurred actively through nuclear pore complexes becauseimport was inhibited by substituting ATP or GTP with ATPγS, AMP-PNP, orGTPγS in the extract, or by functional inhibition of nuclear pores withmAb414, an antibody against several nucleoporins obtained from M. Rout,Rockefeller University, New York (FIG. 8B, SE+414) (Davies and Blobel,Cell 45:699-709, 1986). The ubiquitous transcription factor Oct-1 wasdetected in similar amounts in input nuclei and nuclei exposed tostimulated extract or unstimulated extract (FIG. 8B, Oct-1). Incubationof the nuclei in the unstimulated extract had negligible effect on thelevel of these transcription factors.

Additionally, NFAT, c-Jun/AP1, and NFκB levels were increased in nucleifrom Reh B-cells, 293T fibroblasts, and HeLa cells after incubation inthe stimulated T-cell extract (FIG. 6A). For example, immunologicalanalyses of purified. 293T fibroblast nuclei showed that the stimulatedT-cell extract, but not the control unstimulated T-cell extract,supported nuclear uptake of NFAT, NFκB, and assembly of the AP-1transcription complex (FIGS. 11A and 11B). Notably, the unstimulatedextract supported nuclear import of BSA conjugated to nuclearlocalization signals in resting T-cell nuclei and fibroblast nuclei tothe same extent as the stimulated extract (FIGS. 8B BSA-NLS, and 11B),demonstrating specificity of import and assembly of transcriptionalactivators of the IL-2 gene for the stimulated extract.

For T-cell nuclei exposed to either the stimulated or unstimulatedextract, DNA-binding by these transcription factors was assessed using astandard nuclear retention assay. This assay involves extraction ofnuclei with 0.1% Triton X-100 to dissolve the nuclear membrane andsedimentation at 15,000×g or extraction with 0.5% Triton X-100 for onehour and sedimentation at 10,000×g for 10 minutes (Zhao et al., Cell95:625-636, 1998). Soluble chromatin fractions were prepared frompurified nuclei by micrococcal nuclease digestion and EDTA extraction(O'Neill and Turner, Methods Enzymol. 274:189-197, 1996). Nuclearmatrices, defined as Triton X-100, DNAse, and RNAse extraction-resistantstructures were isolated as described previously (Steen et al., J. CellBiol. 149:531-536, 2000). Immunoblot analysis was performed on thepellet, which contains transcription factors that are bound to DNA, andthe supernatant, which contains the unbound transcription factors. Inparticular, insoluble material was dissolved in SDS, and proteins in thesoluble fraction were precipitated and dissolved in SDS. Equal proteinamounts of both fractions (30 μg) were analyzed by immunoblotting. Thepercentage of DNA-bound transcription factors were determined bydensitometric analysis of duplicate blots. The data is reported as themean±the standard deviation (FIG. 3C).

The results of this nuclear retention assay also support the increasednuclear import and DNA-binding of NFAT, c-Jun/AP1, NFκB, and MAP kinasetranscription factors in reprogrammed T-cell nuclei. For example, anincrease of up to 8.5-fold in intranuclear bound NFAT, AP-1, and NFκBwas detected in nuclei exposed to the stimulated extract compared tonuclei exposed to the unstimulated extract (FIG. 8C). Bound Oct-1 wasdetected in nuclei exposed to unstimulated extract or stimulated extract(FIG. 8C) consistent with its DNA-binding ability in T-cell andnon-T-cell nuclei. A two-fold increase in bound Erk also occurred innuclei exposed to stimulated extract (FIG. 8C). Immunoblotting ofsoluble chromatin and nuclear matrix fractions prepared from nucleiexposed to stimulated extract indicated that NFAT, AP-1, NFκB, and Oct-1were primarily bound to chromatin, whereas most of insoluble Erk wasassociated with the matrix (FIG. 8D).

For demonstration of the effect of exposing T-cell nuclei to thestimulated extract on the DNA-binding of the chromatin remodelingSWI/SNF complex, resting T-cell nuclei were incubated in cell lysisbuffer, the unstimulated extract, or the stimulated extract, eachcontaining an ATP-generating system, for 30 minutes and sedimentedthrough sucrose. The percentage of DNA-bound and unbound human SWI/SNFafter various incubation times was assessed using the above nuclearretention assay with anti-BRG1 antibodies to visualize the SWI/SNFcomplex (FIGS. 4B, 4C, 9A) (Collas et al., J. Cell Biol. 147:1167-1180,1999). In particular, BRG1 was immunoprecipitated from micrococcalnuclease-soluble chromatin pre-cleared with rabbit IgGs using a 1:40dilution of anti-BRG1 antibodies for 2.5 hours. The immune complex wasprecipitated using protein A-sepharose beads, washed inimmunoprecipitation buffer, and dissolved in SDS sample buffer (Collaset al., J. Cell Biol. 147:1167-1180, 1999). Exposure of the nuclei tothe stimulated extract increased the amount of DNA-bound SWI/SNF,suggesting that reprogramming of the nuclei was occurring. For example,densitometric analysis of blots using antibodies to BRG1, a marker ofthe SWI/SNF complex (Zhao et al., Cell 95:625-636, 1998), showed thatover 80% of SWI/SNF was in an insoluble (bound) form in nuclei exposedto the stimulated extract, while SWI/SNF remained mostly soluble ininput nuclei or nuclei exposed to unstimulated extract (FIGS. 9A and9B). Intranuclear binding of SWI/SNF took place within 30 minutes (FIG.9B). The physiological relevance of SWI/SNF binding in vitro wasillustrated by intranuclear anchoring of SWI/SNF within 30 minutes ofanti-CD3 stimulation of human peripheral blood T-cells (FIG. 9C).

The potential activity of SWI/SNF was evaluated by measuring itsrelative ATPase activity in input nuclei and nuclei exposed tostimulated extract or unstimulated extract. Similar amounts of SWI/SNFwere immunoprecipitated from purified nuclear lysates using anti-BRG1antibodies (FIG. 9D). Hydrolysis of 1 nM exogenous ATP by each immuneprecipitate (“BRG1-IP”) was determined in a luciferin-luciferase assay.Control precipitates using pre-immune IgGs were used as a reference(FIG. 9D, Pre-I IgG). ATPase activity was expressed as relative lightunits in the assay after subtraction of the pre-immune IgG referencevalue of 2,700. BRG1-IP purified from input nuclei or nuclei exposed tounstimulated extract displayed no or little ATPase activity. However,BRG1-IP isolated from nuclei exposed to stimulated extract showed an8-fold increase in ATPase activity compared to input nuclei (FIG. 9D).Furthermore, stimulated extract-induced ATPase activity was reducedclose to basal levels when BRG1-IP was treated with DNAse I prior to theassay (FIG. 9D). These results indicate that intranuclear bound SWI/SNFcomplex exhibits DNA-dependent ATPase activity specific for thestimulated T-cell extract.

To measure hyperacetylation of the IL-2 gene of T-cell nuclei in vivoand in vitro, micrococcal nuclease was used to digest the chromatin fromresting T-cells, anti-CD3-stimulated T-cells, T-cell nuclei exposed toan unstimulated extract, and T-cell nuclei exposed to a stimulatedextract, forming soluble chromatin fragments. Acetylated histone H4(“H4ac”) was immunoprecipitated from the soluble chromatin fraction, andDNA was isolated from immune precipitate (“bound”) and supernatant(“unbound”) fractions. The DNA was dot-blotted on duplicate Hybon Nfilters and hybridized to either a fluoresceinated IL-2 probe to theIL-2 coding region or a carp β-actin probe. Hybridization was detectedusing alkaline phosphatase-conjugated anti-fluorescein antibodies.Hyperacetylation of the IL-2 gene, but not the control β-actin gene, wasobserved in nuclei exposed to the stimulated extract, further suggestingthat the nuclei were being reprogrammed to express genes usuallyrepressed by the nuclei.

Acetylation of histone H4 in the IL-2 promoter after T-cell stimulationin culture and in quiescent T-cell nuclei exposed to unstimulatedextract or stimulated extract was also measured by chromatinimmunoprecipitation analysis, using anti-H4 and H4ac antibodies fromSerotec. Hyperacetylation of the IL-2 promoter was examined by chromatinimmunoprecipitation from mock (H₂O) and anti-CD3-stimulated T-cellsafter solubilization with 0.1 U microccocal nuclease per μg DNA to frommono- and di-nucleosomes. An anti-pan-acetylated histone H4 (acH4)antibody was used to detect acetylated histones (O'Neill and Turner,Methods Enzymol. 274:189-197, 1996). DNA was isolated byphenol-chloroform extraction from input, antibody-bound, and unboundchromatin fractions, and IL-2 was identified by dot blot analysis usingan IL-2 probe. The IL-2 promoter probe was synthesized by random primelabeling with fluoresceinated nucleotides (Gene Images CDP-Star,Amersham), using as the template a cloned 430 base pair insertcorresponding to the 360 base pairs of the promoter/enhancer regionsproximal to the start site and the first 70 base pairs of the IL-2coding region (exon I). Primers used were 5′-GCTATTCACATGTTCAGTGTAG-3′(SEQ ID NO: 1) to hybridize the promoter region and5′-GACAGGAGTTGCATCCTGTACA-3′ (SEQ ID NO: 2) to hybridize to exon I. Theβ-actin probe was synthesized as described above using a clonedSalI-NcoI 1.3-kb insert of β-actin intron I as a template (Collas etal., J. Cell Sci. 112:1045-1054, 1999b). Hybridization was detected bychemiluminescence using alkaline phosphatase-conjugated anti-fluoresceinantibodies (Collas et al., J. Cell Sci. 112:1045-1054, 1999b).

In unstimulated T-cells, the IL-2 promoter was entirely detected in theanti-acH4 unbound fraction, suggesting hypoacetylation or absence of H4acetylation of the IL-2 promoter (FIG. 9E, “Culture”). T-cellstimulation, however, elicited hyperactetylation of the IL-2 promoter,as demonstrated by its high enrichment in the anti-acH4-bound fraction(FIG. 9E, “Culture”). Significantly, the IL-2 promoter was also highlyenriched in H4 hyperacetylated chromatin after incubation of nuclei instimulated extract, but not in unstimulated extract (FIG. 9E, “Invitro”). These results are in agreement with reports showing thatchromatin configuration changes occurring in the IL-2 promoter uponT-cell activation are confined to the minimal enhancer region from −300base pairs to the transcription start codon (Ward et al., Nucleic.Acids. Res. 26:2923-2934, 1998; Rao et al., J. Immunol. 167:4494-4503,2001). Altogether, these data provide strong evidence for chromatinremodeling of the IL-2 proximal promoter region in resting T-cell nucleiexposed to the stimulated T-cell extract.

To demonstrate the ability of the stimulated extract to induceexpression of IL-2, resting T-cell nuclei were incubated for 30 minutesat 30° C. in unstimulated extract or stimulated extract. As controls,nuclei were incubated in stimulated extract containing either 100 μg/mlRNAse A, 100 μg/ml DNAse I, mAb414 antibodies, or the lectin WGA. After30 minutes at 30° C., nuclei were lysed in the extracts by sonicationand 3 μl extract aliquots were removed for RT-PCR analysis usingIL-2-specific primers. In particular, total RNA was isolated using theQiagen RNeasy kit, and 15 ng RNA was used as the template for RT-PCRusing the Promega Access RT-PCR System. A 467-bp IL-2 cDNA was amplifiedusing the IL-2-specific primers 5′-ATGTACAGGATGCAACTCCT GTCTT-3′ (SEQ IDNO: 3) and 5′-GTTAGTGTTGAGATGATGCTTTGAC-3′ (SEQ ID NO:4). PCR conditionswere 30 cycles of denaturation at 94° C. for one minute, annealing at60° C. for two minutes, and extension at 72° C. for three minutes. Inputstimulated extract and a control stimulated extract containing 1.2 μgtotal RNA isolated from IL-2-producing T-cells were also analyzed (FIGS.5A and 10A). These results indicate that IL-2 expression was induced byincubation of T-cell nuclei in the stimulated extract but not byincubation in any of the control extracts. The PCR product of theexpected size (467 base pairs) was absent from input nuclei and inputstimulated extract (as expected from FIG. 7), nuclei exposed tounstimulated extract (Nuclei-USE), and nuclei exposed to stimulatedextract followed by treatment with 100 μg/ml RNAse A, but not 100 μg/mlDNAse I, prior to RT-PCR. Thus, detection of IL-2 mRNA was the result ofIL-2 transcription and not of RNA contamination in input nuclei or inthe extract. IL-2 transcription required active nuclear import becauseIL-2 transcription was abolished when nuclear pore function was blockedin the stimulated extract with the mAb414 antibody or 0.5 mg/ml wheatgerm agglutinin (FIG. 10A).

As illustrated in FIGS. 5B and 10B, this in vitro IL-2 mRNA productionis dependent on PolII transcription. For this assay, nuclei were exposedfor 30 minutes to stimulated extract containing increasingconcentrations of the RNA PolII inhibitor actinomycin D (0, 5, 10, 50,100 and 500 nM), and IL-2 mRNA synthesis was analyzed by RT-PCR. As acontrol, extracts from anti-CD3 stimulated B-cells, fibroblasts, andHeLa cells, which do not express IL-2, were tested for their ability toinduce IL-2 expression in nuclei from resting T-cells. As expected,these extract failed to induce IL-2 expression. Arrows in FIGS. 5A-5Cpoint to the 458 base pair IL-2 RT-PCR product.

As a more stringent indicator of nuclear reprogramming, activation ofthe IL-2 gene was monitored in nuclei purified from primary humanumbilical vein endothelial cells (HUVEC), NT2 neuronal precursors, andHeLa cells after a two hour incubation in the stimulated extract. RT-PCRanalysis indicated that the stimulated extract activated the IL-2 genein the nuclei of all of these cell types; in contrast, the unstimulatedextract was ineffective at inducing IL-2 transcription (FIG. 10C). IL-2activation was dependent on RNA Pol II activity and nuclear import,based on its elimination by 50 nM actinomycin D and mAb414, respectively(FIG. 10C). Lastly, the specificity of IL-2 induction for the stimulatedextract was demonstrated by IL-2 remaining repressed in resting T-cellnuclei exposed to control extracts from 293T fibroblasts, HeLaendothelial cells, or Bjab B-cells that had been treated with anti-CD3and cross-linking antibodies to mimic T-cell stimulation (FIG. 10D).

In summary, these results demonstrate that nuclear reprogramming, asevidenced by transcriptional activation of a silent gene, can be inducedin purified intact nuclei. Expression of the IL-2 gene was coincidentwith physiological nuclear uptake and assembly of transcriptionalregulatory proteins. It is noteworthy that NFAT, NFκB, and AP-1 aretranscription factors that reflect a proliferative response rather thana differentiation response per se. Remodeling of chromatin wasdemonstrated by intranuclear anchoring and DNA-dependent ATP hydrolysisactivity of the SWI/SNF complex. The SWI/SNF complex uses energy of ATPhydrolysis to alter nucleosomal conformation. Notably, the stimulatedextract elicited an 8-fold enhancement of ATPase activity over that ofinput resting T-cell nuclei, from equivalent amounts ofimmunoprecipitated BRG1. Thus, increased ATPase activity is the resultof activation of SWI/SNF rather than a consequence of higher amounts ofprecipitated BRG1 in nuclei exposed to stimulated extract. Histone H4hyperacetylation of the IL-2 proximal promoter region, which involves acomplex targeting acetyltransferases to their site of action, furtherindicates active chromatin remodeling. While not meant to limit theinvention to any particular mechanism, H4 acetylation in the IL-2promoter may stimulate binding of transcriptional activators that wouldotherwise be excluded from repressed chromatin, or transcription factorbinding may promote alterations in chromatin structure. The resultsdescribed herein indicate nuclear reprogramming can take place in intactnuclei in vitro. The results also demonstrate that the process involvesthe active intranuclear assembly of protein complexes that remodelchromatin as well as the binding of transcriptional regulators.

Example 5 Reprogramming of Fibroblasts Using an Activated T-Cell Extract

As demonstrated in Example 4, a stimulated T-cell extract increasednuclear localization of T-cell specific transcription factors in 293Tfibroblasts. The ability of fibroblasts to be reprogrammed into T-cellsis characterized further below.

For this study, T-cells were purified from peripheral blood from healthydonors, as described in Example 4 (Skalhegg et al., Science 263:84-87,1994). To prepare reprogramming extracts from stimulated T-cells, cellswere frozen in liquid nitrogen at 5-10 minutes post-stimulation, thawed,washed in ice-cold lysis buffer (Collas et al., J. Cell Bio.147:1167-1180, 1999), and sedimented at 400×g. The pellets wereresuspended in two volumes of lysis buffer. Cells and nuclei weredisrupted with a tip sonicator, and the lysate was cleared bycentrifugation at 15,000×g for 15 minutes at 4° C. The supernatant wasused immediately or was frozen in liquid nitrogen and stored at −80° C.Unstimulated T-cell extracts were prepared from mock (H₂O)-stimulatedT-cells.

Nuclear reprogramming reactions consisted of 20 μl or multiples thereofof stimulated T-cell extract or unstimulated T-cell extract containing100,000 nuclei and an ATP generating system (1 mM ATP, 10 mM creatinephosphate, 25 μg/ml creatine kinase, and 100 μM GTP). Reactions wereincubated at 30° C. for 30 minutes unless indicated otherwise. At theend of incubation, nuclei were purified by sedimentation through 1 Msucrose. Alternatively, total RNA was extracted from the reaction mixfor RT-PCR.

The active uptake of transcription factors in fibroblast nuclei exposedto the stimulated T-cell extract was further demonstrated by the abilityof a monoclonal antibody that reacts with nucleoporins and inhibitsnuclear pore function (obtained from M. Rout, Rockefeller University,New York, N.Y., USA) to reduce nuclear import of these factors (FIG.11B, mAb414). Oct-1 was detected in 293T input nuclei and nuclei exposedto stimulated T-cell extract or control unstimulated T-cell extract(FIG. 11B), consistent with its DNA-binding property in several celltypes. Immunoblotting of chromatin and nuclear matrices of 293T nucleitreated with the stimulated T-cell extract indicated that NFAT, AP-1,NFκB, and Oct-1 were primarily bound to chromatin (FIG. 11C).Altogether, these data demonstrate physiological uptake oftranscriptional regulators by the fibroblast nuclei from the extract.

Intranuclear anchoring of the human nucleosome remodeling complexSWI/SNF was also investigated. Anti-CD3 stimulation of T-cells elicitedintranuclear anchoring of the SWI/SNF complex, as determined byimmunoblotting of Triton X-100-soluble and insoluble nuclear fractionswith an antibody against BRG1, a marker of the SWI/SNF complex (FIG.12A). In particular, BRG1 was immunoprecipitated from micrococcalnuclease (MNase)-soluble chromatin pre-cleared with rabbit IgGs, usinganti-BRG1 antibodies (dilunstimulated T-cell extract 1:40) for 2.5hours. The immune complex was precipitated using protein A-sepharosebeads, washed three times in immunoprecipitation buffer (Collas et al.,J. Cell Bio. 147:1167-1180, 1999), and dissolved in SDS sample buffer.Western blots were performed using antibody dilutions of 1:500 (Collaset al., J. Cell Bio. 147:1167-1180, 1999). Over 80% of SWI/SNF wasdetected in a bound form within 20 minutes in stimulated T-cellextract-treated 293T nuclei; in contrast, SWI/SNF remained soluble innuclei exposed to unstimulated T-cell extract (FIG. 12A).

Additionally, ATPase activity of the SWI/SNF complex was determined in astandard luciferin-luciferase assay after immunoprecipitation of thecomplex using anti-BRG1 antibodies. BRG1 immune precipitates(“BRG1-IPs”) purified from 293T input nuclei or unstimulated T-cellextract-treated nuclei displayed no or little ATPase, based on elevatedATP levels in the assay (FIG. 12B). However, BRG1-IP isolated fromnuclei treated with the stimulated T-cell extract displayed an ˜8-foldincrease in ATPase activity, reducing the ATP level in the assay from2,500 to 300 RLU. No activity was detected in control precipitates usingpre-immune IgGs (FIG. 12B). These results indicate that the stimulatedT-cell extract promotes intranuclear anchoring of the SWI/SNF nucleosomeremodeling complex and ATPase activity of the bound complex in 293Tnuclei.

Potential for gene expression often correlates with hyperacetylation ofhistone H4. As an additional marker of nuclear reprogramming, changes inH4 acetylation at the IL-2 locus in stimulated T-cell extract- andunstimulated T-cell extract-treated 293T nuclei were measured. Chromatinimmunoprecipitation (ChIP) experiments were performed using an antibodyagainst all forms of acetylated H4 (“acH4,” FIG. 12C). In particular,intact nuclei were isolated from 293T, NT2, and unstimulated peripheralblood T-cells by Dounce-homogenization and stored frozen (Collas et al.,J. Cell Bio. 147:1167-1180, 1999). Soluble chromatin was prepared frompurified nuclei by MNase digestion (O'Neill and Turner, Methods Enzymol.274:189-197, 1996), and nuclear matrices, defined as Triton X-100,DNAse, and RNAse extraction-resistant structures, were isolated asdescribed (Steen et al., J. Cell Biol. 149:531-536, 2000). ChIP wasperformed after solubilization of chromatin with 0.1 U MNase per μg DNAusing an anti-pan-acetylated histone H4 antibody (O'Neill and Turner,Methods Enzymol. 274:189-197, 1996). DNA was isolated byphenol-chloroform extraction from antibody-bound and unbound fractions,and the IL-2 locus was identified by dot blot analysis. An IL-2 probewas synthesized and fluoresceinated by random priming (Amersham) usingas a template a 467-bp IL-2 PCR product amplified from genomic DNA byPCR as described above. Hybridization was detected by chemiluminescence(Collas et al., J. Cell Sci. 112:1045-1054, 1999). The β-actin probe wassynthesized and labeled as described (Collas et al., J. Cell Sci.112:1045-1054, 1999).

In input nuclei and nuclei incubated in unstimulated T-cell extract,IL-2 was detected exclusively in anti-acH4 unbound chromatin, suggestinghypoacetylation of H4 in the IL-2 locus (FIG. 12C). In contrast, IL-2was detected in anti-acH4 bound chromatin of nuclei treated withstimulated T-cell extract, reflecting enhanced H4 acetylation at theIL-2 locus in these nuclei (FIG. 12C). As anticipated, reprobing filterswith a probe against the constitutively expressed β-actin gene revealedhyperacetylated H4 at the β-actin locus (FIG. 12C). Thus, stimulatedT-cell extract elicits enhanced H4 acetylation at the IL-2 locus in 293Tnuclei, providing evidence for physiological chromatin remodeling.

Another and more stringent indicator of nuclear reprogramming wasinduction of IL-2 transcription in 293T fibroblast nuclei exposed tostimulated T-cell extract (FIG. 12D). Total RNA was isolated using theQiagen RNeasy kit, and 15 ng RNA was used as the template for RT-PCRusing the Promega Access RT-PCR System. A 467-bp product was amplifiedusing the IL-2-specific primers 5′-ATGTACAGGATGCAACTCCTGTCTT-3′ (SEQ IDNO: 5) and 5′-GTTAGTGTTGAGATGATGCTTTGAC-3′ (SEQ ID NO: 6) by 30 cyclesof denaturation at 94° C. for 30 seconds, annealing at 60° C. for oneminute, and extension at 72° C. for one minute. RT-PCR analysisindicated that the IL-2 gene was activated in the stimulated T-cellextract, but not in the unstimulated T-cell extract. As expected fromthe above results, the IL-2 transcript was absent from input nuclei,input stimulated T-cell extract, and nuclei exposed to stimulated T-cellextract containing either 100 μg/ml RNAse A, mAb414, or 50 nM of the RNApolymerase II (Pol II) inhibitor, actinomycin D (FIG. 12D). Similarresults were obtained with neuronal precursor NT2 nuclei and restingT-cell nuclei (FIG. 12D). Collectively, these data indicate that thestimulated T-cell extract supports chromatin remodeling and RNA PolII-dependent activation of the repressed IL-2 gene in T-cell andnon-T-cell nuclei.

Example 6 Reprogramming of Permeabilized Cells

The ability to reprogram whole cells, in addition to purified nuclei,was demonstrated as described below. 293T fibroblasts grown oncoverslips were reversibly permeabilized with the bacterial toxinStreptolysin O, exposed to extract of readily available stimulatedJurkat cells or neuronal precursor cells, resealed with 2 mM CaCl₂, andexpanded in culture. Reprogramming into T-cells was evaluated byalterations in gene expression, expression of T-cell-specific proteins,and induction of a T-cell-specific function in the reprogrammed 293Tfibroblasts. Reprogrammed 293T fibroblasts exposed to neuronal extractswere analyzed for expression of neuronal proteins.

For these studies, 293T fibroblasts were grown on 16-mmpoly-L-lysine-coated coverslips in RPMI1640 to 100,000 cells/coverslipin 12-well plates. Cells were permeabilized in 200 ng/ml streptolysin Oin Ca²⁺-free Hanks Balanced Salt Solution (Gibco-BRL) for 50 minutes at37° C. in regular atmosphere. Over 80% of 293T-cells were permeabilizedunder these conditions, as judged by propidium iodide uptake.Streptolysin O was aspirated; coverslips overlaid with 80 μl of either293T, Jurkat-Tag, or NT2 extract; and incubated for one hour at 37° C.in CO₂ atmosphere. Each extract contained the ATP generating system and1 mM each of ATP, CTP, GTP and UTP. Extracts from Jurkat-TAg cells wereprepared as described above after co-stimulation for 1-2 hours with 40ng/ml anti-CD3 antibodies (clone SpvT3d obtained from A. M. Rasmussen,Norwegian Radium Hospital, Montebello, Norway) and 0.1 μM PMA. Theneuronal precursor NT2 extract was prepared from confluent NT2 cells(Stratagene) by sonication and sedimentation as described above. Toreseal plasma membranes, RPMI1640 containing 2 mM CaCl₂ (added from a 1M stock in H₂O) was added to the wells, and the cells were incubated fortwo hours at 37° C. This procedure resealed ˜100% of the permeabilizedcells. Ca²⁺-containing RPMI was replaced by RPMI, and the cells wereexpanded for several weeks.

Transcription levels in reprogrammed fibroblasts exposed to the Jurkatextract were compared to those of 293T-cells exposed to a 293T extract(‘control cells’) 10 days post-reprogramming reaction. A human cytokineexpression array containing 375 cDNAs was used to monitor changes ingene expression. In particular, mRNA was isolated (mRNA Direct™, Dynal)from ‘reprogrammed’ and control cell pellets frozen in liquid nitrogenat 10 days post-reprogramming. One μg mRNA was used as the template forcDNA synthesis (cDNA Labeling and Hybridization Kit, R&D Systems) withα³³P-dCTP and cytokine-specific primers (R&D Systems) according to themanufacturer's protocol. Purified probes were hybridized to HumanCytokine Expression Arrays (R&D Systems) under recommended conditions.Arrays were exposed to a phosphorscreen for six days. Hybridization wasquantified on a phosphorimager and analyzed using the Phoretix Array V.2analysis software.

Over 120 genes were up- or down-regulated as a result of reprogramming(FIG. 13; only transcripts up- or down-regulated more than two-fold areshown). Subsets of genes encoding hematopoietic cell surface antigens,interleukins and interleukin receptors, cytokines and cytokinereceptors, chemokines and chemokine receptors, epidermal growth factors,and orphan receptors were up-regulated. Several genes of the FGF,adhesion molecule, and integrin families were down-regulated. Severalgenes of the TGFβ and TNF families were also either up- ordown-regulated. No neutrophic factor transcripts were affected, nor wasexpression of house keeping genes affected (FIG. 13). Similar resultswere obtained in duplicate arrays from separate reactions examined at 13days post-reprogramming. Thus, hematopoietic genes are turned on orup-regulated in 293T-cells exposed to a Jurkat extract, whereas genesfor FGFs, adhesion molecules, and cytoskeletal components aredown-regulated or repressed.

Expression of hematopoietic cell-specific surface antigens inreprogrammed fibroblasts was also evaluated. Immunofluorescence analysisof IL-2Rα and β was performed as described (Collas et al., J. Cell Bio.147:1167-1180, 1999; anti-IL-2Rα and IL-2Rβ antibodies were obtainedfrom R&D Systems). Analysis of other surface antigens was performedusing FITC- or TRITC-conjugated primary antibodies (FITC-conjugatedanti-CD3, CD4, CD8, and CD45 antibodies from Diatec and FITC-conjugatedanti-TCRαβ antibody from Pharmingen). Immunofluorescence analysis showedthat CD3, CD4 and CD8 were detected by 4 days post-reprogramming, andthe CD45 tyrosine phosphatase was detected by 11 days post-reprogrammingin most reprogrammed cells but not in control cells (FIG. 14A).Furthermore, the α and β chains of the TCR complex were expressed in thereprogrammed fibroblasts, based on immunofluorescence labeling with anantibody against TCRαβ (FIG. 14B).

Expression of immune cell surface receptors in reprogrammed fibroblastsprompted investigation of functional reprogramming. Unstimulated T-cellsexpress the low affinity IL-2 receptor β (IL-2Rβ). High affinity IL-2Rrequires induction of IL-2Rα by TCR-CD3 complex stimulation.TCR-CD3-dependent induction of IL-2Rα is indicative of normal TCRfunction. FIG. 14C illustrates that reprogrammed fibroblasts expressedIL-2Rβ, but not IL-2Rα, in the absence of stimulation. Furthermore, inreprogrammed cells, anti-CD3 and phorbolmyristylacetate (PMA)stimulation elicited expression of IL-2Rα that co-localized with IL-2Rβ,as shown in overlay images (FIG. 14C, “+Stimulation”). Similar resultswere observed with the Jurkat cells that were used to prepare theextract (FIG. 14C). As expected, stimulation of control fibroblasts didnot significantly induce IL-2Rα. Altogether, these results indicate theexpression of functional immune-specific receptors in the reprogrammedcells.

To demonstrate the general applicability of in vitro cell reprogramming,permeabilized fibroblasts were exposed to an NT2 cytoplasmic and nuclearextract for one hour at 37° C. as described above for Jurkat extracts.The cells were resealed and cultured for 15 days in low confluency inRPMI1640. Then, the expression of neurofilament protein NF200 (Debus etal., Differentiation 25:193-203, 1983) was examined byimmunofluorescence. NF200 was strongly expressed in fibroblasts exposedto the NT2 extract, but not to a control 293T fibroblast extract (FIG.15). Furthermore, NF200 appeared restricted to polarized outgrowths fromthe fibroblasts resembling elongating neurites, which occasionallycontacted neighboring cells in culture. These data indicate thatneuron-specific proteins can be expressed in fibroblasts under thesereprogramming conditions.

In summary, these results demonstrate functional reprogramming of asomatic cell using a nuclear and cytoplasmic extract derived fromanother somatic cell type. These experiments illustrate activation ofrepressed genes and synthesis of proteins specific for another cell typein somatic fibroblasts by exposure to extracts from heterologous somaticcell types. Reprogramming fibroblast genome function in a T-cell orJurkat-TAg extract is demonstrated by physiological nuclear uptake andassembly of transcriptional regulatory proteins, chromatin remodeling,activation of lymphoid-specific genes, down-regulation of selective setsof genes, expression of T-cell-specific antigens including CD3 and TCR,and establishment of the IL-2R assembly pathway in response to CD3-TCRstimulation. Moreover, a neurofilament protein was expressed infibroblasts exposed to a neuronal precursor cell extract. In vitroreprogramming of differentiated somatic cells from primary culturescreates a wide range of possibilities to produce isogenic orsubstantially isogenic replacement cells for therapeutic applications.

Example 7 Reprogramming to Generate Stem Cells

An embryonic stem cells extract was used to reprogram permeabilized,mouse fibroblasts as described below. Similar methods can be used toreprogram other cells, such as other fibroblasts (e.g., human skinfibroblasts).

Briefly, mouse embryonic stem cells were cultured in the presence ofleukemia inhibitory factor (LIF) without feeder layers using standardprocedures. An embryonic stem cell reprogramming extract was prepared asfollows. Embryonic stem cells were harvested, washed three times in PBS,washed once in ice-cold cell lysis buffer described above, and the cellpellet was resuspended in an equal volume of cell lysis buffer. Thesuspension was sonicated on ice until all cells and nuclei weredisrupted. The resulting lysate was centrifuged at 15,000×g for 15minutes at 4° C. The supernatant (“reprogramming extract”) was eitherused fresh or aliquoted, snap-frozen in liquid nitrogen, and stored at−80° C. until use.

Mouse NIH3T3 cells, a transformed fibroblast cell line, were grown onto12-mm round glass coverlips coated with poly-L-lysine to a density of50,000 cells per coverslip. Cells were permeabilized with Streptolysin Oas described above. Sreptolysin O was removed by gentle aspiration andreplaced by 80 μg of mouse embryonic stem cell extract containing an ATPgenerating system and nucleotides. Cells were incubated in the extractfor one hour at 37° C. in regular atmosphere. Culture medium (500 μl)containing 2 mM CaCl₂ was added directly to the cells which weresubsequently allowed to reseal for two hours at 37° C. in a CO₂incubator. CaCl₂-containing medium was removed and replaced by regularembryonic stem culture medium containing LIF.

Reprogrammed NIH3T3 cells were cultured and examined on day fourpost-reprogramming. Phase contrast microscopy analysis showed that thecells grew in clumps, forming ‘colonies’ resembling those formed byembryonic stem cells (compare FIG. 16A and FIG. 16B). Some of the largercolonies such as that shown on FIG. 16B lifted off the culture dish toform embryoid bodies. In contrast, control fibroblasts permeabilizedwith Streptolysin O and exposed to a control NIH3T3 extract did not formcolonies and maintained a typical fibroblast phenotype (compare FIG. 16Bwith input NIH3T3 cells in FIG. 16A). Similarly, control intact(non-permeablizied) NIH3T3 cells exposed to the embryonic cell extractdid not acquire the embryonic cell phenotype (FIG. 16B). Embryonic stemcell morphology of the reprogrammed cells was seen for at least 10 daysin culture.

As a molecular marker of reprogramming, the reprogrammed cells wereexamined for the expression of Oct4, the product of the Oct4 gene. Oct4expression is unique to germ cells, stem cells, preimplantation embryos,and the epiblast of the early post-implantation embryos. Therefore, Oct4expression represents a useful marker for identification of pluri- ortoti-potent cells. Oct4 expression was monitored four days afterreprogramming by immunofluorescence using a commercially availableanti-Oct4 antibody (Santa Cruz Biotechnology). FIG. 17A shows a clearanti-Oct4 labeling in input embryonic stem cells. As expected, NIH3T3fibroblasts were not labeled (FIG. 17A). NIH3T3 cells reprogrammed inthe embryonic stem cell extract exhibited anti-Oct4 labeling; incontrast, NIH3T3 cells exposed to a control NIH3T3 extract or intactNIH3T3 cells exposed to the embryonic stem cell extract (FIG. 17A) werenot labeled with anti-Oct4. This result indicates that the reprogrammedcells express the embryonic stem cell-specific transcription factor,Oct4.

Immunofluorescence observations were verified by Western blottinganalysis. FIG. 17B shows that while input NIH3T3 cells did not expressOct4 (“NIH”), NIH3T3 cells exposed to the embryonic stem cell extractexpressed high levels of Oct4 (“NIH/ES ext.”). The expression level wassimilar to that of embryonic stem cells used to prepare the extract(FIG. 17B, “ES”). Control NIH3T3 cells exposed to NIH3T3 cell extractdid not express Oct4, as anticipated (FIG. 17B). 50,000 NIH3T3 cells and10,000 embryonic stem cells and reprogrammed cells were used in theimmunoblot shown in FIG. 17B.

A novel, rapid, sensitive and semi-quantitative assay was developed tomeasure alkaline phosphatase, another embryonic stem cell marker, inembryonic stem cells and in the reprogrammed cells. The assay is basedon spotting 1-2 μl of a Triton X-100 soluble lysate of embryonic stemcells, NIH3T3 cells exposed to NIH3T3 extract, intact NIH3T3 cellsexposed to embryonic stem cell extract, or of any cell of choice on adry nitrocellulose membrane or on any other appropriate solid support.The test spot is of known protein concentration or from a known cellnumber to allow comparison to other spots. If desired, an aliquotcontaining a known amount of alkaline phosphatase or having a knownlevel of alkaline phosphatase activity can also be spotted on the filterto form a reference spot. The membrane was wetted in Tris-bufferedsaline and drained. Alkaline phosphatase was detected by applying adetection solution normally designed to detect alkalinephosphatase-conjugated DNA probes on Southern blots (Alk-Phos Directdetection solution, Amersham). Alkaline phosphatase dephosphorylates asubstrate contained in the detection solution, resulting in lightemission. The membrane was drained and exposed to film. If the testsample is applied to a solid support other than a membrane, such as a96-well plate, than either the 96-well plate is exposed to film or, itis exposed to a CCD camara to measure light emitted by the alkalinephosphate detection reaction. Alkaline phosphatase in the embryonic stemcell lysate, but not in the NIH3T3 cell lysate, resulted in theappearance of a light spot detected on the film (FIG. 18). Additionally,permeabilized NIH3T3 cells reprogrammed in embryonic stem cell extract,but not control cells, had alkaline phosphatase (FIG. 19).

The amount of alkaline phosphatase in the test spot can be determined bycomparing the signal from the test spot to the signal from the referencespot or to the signal from a series of reference spots with increasinglevels of alkaline phosphatase (e.g., forming a standard curve). Theconcentration of protein or the number of cells used to derive the testspot (e.g., the units of alkaline phosphatase mg protein) can be used toextrapolate the level of alkaline phosphatase in the original test cellor sample.

The reprogrammed cells were passaged and replated on day four usingstandard embryonic stem culture techniques, using LIF supplementedmedium. Ten days after reprogramming, however, Oct4 expression levelswere greatly reduced in the reprogrammed cells. The cells also lost thetypical embryonic stem cell colony morphology they acquired after thereprogramming reaction. This result may be the result of either (i)transient reprogramming of the cells, i.e., the reprogramming factorsare diluted out as the reprogrammed cells divide and are no longeractive, (ii) spontaneous differentiation of the reprogrammed embryonicstem cells into fibroblasts, or (iii) loss of the truly reprogrammedembryonic stem cells such that contaminating non-reprogrammedfibroblasts outgrew the embryonic stem cells and remained in theculture. If desired, expression of Oct4 and other stem cell-specificproteins may be maintained in the reprogrammed cells for a longer periodof time by performing multiple rounds of reprogramming. Moreover, thepermeabilized cells can be exposed to the stem cell extract for a longerperiod of time during each round of reprogramming. Additional nuclearfactors can also be added to the stem cell extract as described above tomaximize reprogramming.

Collectively, these data indicate that NIH3T3 cells exposed to anembryonic stem cell extract acquire an embryonic stem cell phenotype,express Oct4, and express alkaline phosphatase.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A method of altering gene expression of a mammalian fibroblast, saidmethod comprising incubating a permeabilized fibroblast with anactivated T-cell extract, said permeabilized fibroblast having pores inits plasma membrane or a partial plasma membrane, wherein saidincubation results in an alteration of gene expression in saidfibroblast, wherein said fibroblast with altered gene expressionexpresses IL-2 receptor.
 2. The method of claim 1, wherein thefibroblast with altered gene expression is incubated under conditionsthat allow the membrane of said fibroblast to reseal.
 3. The method ofclaim 1, wherein said fibroblast is permeabilized by incubating anintact fibroblast with a detergent or a bacterial toxin.
 4. The methodof claim 3, wherein said bacterial toxin is Streptolysin O.
 5. Themethod of claim 1, wherein said fibroblast or said T-cell is a humancell.
 6. The method of claim 1, wherein said permeabilized fibroblast isan interphase or mitotic cell.
 7. The method of claim 1, wherein saidalteration in gene expression involves a DNA methyltransferase, histonedeacetylase, histone, nuclear lamin, activator, repressor, growthfactor, hormone, or cytokine.
 8. A method of altering gene expression ofa mammalian fibroblast, said method comprising incubating apermeabilized mammalian fibroblast with an interphase cell extract froman embryonic stem cell, said permeabilized mammalian fibroblast havingpores in its plasma membrane or a partial plasma membrane, wherein saidincubation results in an alteration of gene expression in said mammalianfibroblast, wherein said fibroblast with altered gene expressionexpresses Oct4 or alkaline phosphatase.
 9. The method of claim 8,wherein said mammalian fibroblast or said embryonic stem cell is a humancell.
 10. The method of claim 8, wherein said fibroblast with alteredgene expression forms embryonic stem cell-like colonies.
 11. The methodof claim 8, wherein said fibroblast with altered gene expression formsembryoid bodies.
 12. The method of claim 8, wherein the fibroblast withaltered gene expression is incubated under conditions that allow themembrane of said fibroblast to reseal.
 13. The method of claim 8,wherein said mammalian fibroblast is permeabilized by incubating anintact cell with a detergent or a bacterial toxin.
 14. The method ofclaim 13, wherein said bacterial toxin is Streptolysin O.
 15. A methodof altering gene expression of a mammalian fibroblast, said methodcomprising incubating a permeabilized mammalian fibroblast with aninterphase cell extract from a neural cell, said permeabilized mammalianfibroblast having pores in its plasma membrane or a partial plasmamembrane, wherein said incubation results in an alteration of geneexpression in said mammalian fibroblast, wherein said fibroblast withaltered gene expression expresses neurofilament protein NF200.
 16. Themethod of claim 15, wherein said neural cell is a neuronal precursorcell.
 17. The method of claim 15, wherein said mammalian fibroblast orsaid neural cell is a human cell.
 18. The method of claim 15, whereinsaid fibroblast with altered gene expression forms a neurofilament,neurite, or axon.
 19. The method of claim 15, wherein said fibroblastwith altered gene expression divides or is immortalized.
 20. The methodof claim 15, wherein the fibroblast with altered gene expression isincubated under conditions that allow the membrane of said fibroblast toreseal.
 21. The method of claim 15, wherein said mammalian fibroblast ispermeabilized by incubating an intact cell with a detergent or abacterial toxin.
 22. The method of claim 15, wherein said bacterialtoxin is Streptolysin O.